Nucleic Acid Biology and its Application in Human Diseases 9811985197, 9789811985195

Table of contents :
Preface
Contents
Editors and Contributors
1: Nucleic Acid Structure and Biology
1.1 Introduction
1.2 Nucleoside Conformation and Hydrogen Bonding
1.2.1 Sugar Conformation
1.2.2 Syn/Anti Conformation
1.2.3 Base Pair Arrangements
1.3 Double-Helical Nucleic Acids
1.3.1 A-, B-, and Z-Type Helices
1.3.2 Superhelicity and Superhelical Stress
1.4 Double-Helical Junctions in Nucleic Acids
1.4.1 Three-Way Junctions
1.4.1.1 Three-Way Junctions in Biology and Technology
1.4.2 Four-Way Junctions
1.4.2.1 Holliday Junctions
1.4.2.2 Cruciform Structures
1.4.2.3 Four-Way Junctions in Biology and Technology
1.5 Pseudoknots
1.5.1 Pseudoknots in Biology
1.6 Triple-Helical Structures
1.6.1 Intermolecular Triple Helices
1.6.2 H-DNA
1.6.3 Triplexes in Biology and Technology
1.7 The G-Quadruplex
1.7.1 Architecture of the G-Quadruplex Core
1.7.2 G-Quadruplex Topologies
1.7.3 G-Quadruplexes in Biology and Technology
1.8 The i-Motif
1.8.1 i-Motif Structures in Biology and Technology
1.9 Impact of Base Modifications on DNA Structure
1.10 Summary
References
2: Nucleic Acid-Mediated Inflammatory Diseases
2.1 Introduction
2.2 PRR Receptors
2.2.1 TLRs
2.2.1.1 TLR3
2.2.1.2 TLR9
2.2.1.3 TLR7
2.2.1.4 TLR8
2.2.1.5 TLR10 (in Humans)
2.2.1.6 TLR 13 (in Mouse)
2.2.2 NLRs
2.2.3 RLRs
2.2.4 ALRs
2.2.5 cGAS
2.2.6 Other Receptors
2.3 PRRs and Cell Death
2.3.1 Toll-like Receptors (TLR)
2.3.1.1 TLR9
2.3.1.2 TLR3
RIG-1-like Receptors (RLR)
Absent in Melanoma-2-like Receptors (ALRs)
IFI16
Intracellular DNA Sensors Such as cGAS
ZBP1
2.4 Nucleic Acid Sensors and Inflammatory Diseases
2.5 Autoimmune Disorders
2.5.1 Systemic Lupus Erythematosus (SLE)
2.5.2 Aicardi-Goutieres Syndrome (AGS)
2.5.3 Singleton-Merten Syndrome (SMS)
2.5.4 Rheumatoid Arthritis (RA)
2.5.5 Other Inflammatory Conditions
2.5.6 Nucleic Acid Sensors as Therapeutic Targets
2.5.7 Autoimmune Disorders
2.5.8 Infectious Disease
2.5.9 Cancer
2.6 Discussion
References
3: Alternative Splicing and Cancer
3.1 Introduction
3.2 Mechanisms of Alternative Splicing Regulation
3.3 Alternative Splicing of Non-coding RNAs
3.4 Relation Between Alternative Splicing and lncRNA
3.5 Relation Between Alternative Splicing and miRNA
3.6 Relation Between Alternative Splicing and siRNA
3.7 Relation Between Alternative Splicing and snoRNA
3.8 Dysregulation of Alternative Splicing in Cancer
3.8.1 Mutations in Cis-Regulatory Splice Sites
3.8.2 Mutations in Spliceosomal and Trans-Regulatory Splicing Factors
3.8.3 Alteration in Expression of Trans-Regulatory Splicing Factors
3.8.4 Alteration in Pathways Regulating Splicing Factors
3.9 Role of G-Quadruplex Motif at Splice Sites in the Regulation of Alternative Splicing
3.9.1 Alteration in Post-translational Modifications, Epigenetic Regulation, and Other Regulatory Mechanisms
3.10 Dysregulated Alternative Splicing and Hallmarks of Cancer
3.11 Modulation in Alternative Splicing as a Target for Cancer Detection and Therapeutics
3.11.1 Use of Antisense Oligos (ASOs) or Splicing-Switching Oligos (SSOs)
3.11.2 Use of Small Molecules
3.11.3 Use of Monoclonal Antibodies (mAbs) Targeting Deregulated Proteins
3.11.4 Use of Chemotherapeutic Drugs to Modify Splicing
3.11.5 Use of Spliceosome-Mediated RNA Trans-Splicing (SMaRT) Technique
References
4: Nucleic Acid-Based Strategies to Treat Neurodegenerative Diseases
4.1 Introduction
4.2 Technologies Under Nucleic Acid Therapeutics
4.2.1 Antisense Oligonucleotides (ASO)
4.2.1.1 Ligand-Modified Small Interfering RNA Conjugates
4.2.1.2 Anti-miRNA Oligonucleotides (antagoMIR)
4.2.1.3 Lipid Nanoparticles
4.2.1.4 Adeno-associated Virus Vectors
4.3 Strategies Involved in the Treatment of Neurodegenerative Disorders
4.3.1 Alzheimer´s Disease
4.3.2 Parkinson´s Disease
4.3.3 Huntington´s Disease
4.3.4 Spinal Muscular Atrophy
4.3.5 Frontotemporal Dementia (FTD)
4.3.6 Amyotrophic Lateral Sclerosis
4.4 Conclusion
References
5: Human Diseases Induced by Oxidative Damage in DNA
5.1 Introduction
5.2 Types of Oxidative Damage
5.2.1 Formation of 8-Oxo-G
5.2.2 Formation of 8-Nitro-G
5.3 Oxidative DNA Damage Repair Systems
5.3.1 Base Excision Repair (BER) Pathway
5.3.2 Nucleotide Excision Repair (NER) Pathway
5.4 Oxidative DNA Damage Can Disrupt Cellular Function
5.5 Human Diseases Induced by Oxidative DNA Damage
5.5.1 Oxidative Stress in Cancer Prognosis
5.5.2 In Neurodegenerative Diseases
5.5.3 In Inflammation/Infection
5.5.4 In Cardiovascular Diseases
5.5.5 Aging/Infertility and Metabolic Syndromes
5.5.6 Genetic Diseases Due to DDR Damage
5.6 Treatment of Diseases by Targeting DNA Damage and DDR Pathways
5.7 Conclusion
References
6: Nucleic Acid in Nanotechnology
6.1 Introduction
6.2 Structural DNA-Based Nanotechnologies
6.2.1 DNA Tiles
6.2.2 DNA Bricks
6.2.3 DNA Origami
6.2.4 DNA Crystals
6.2.5 DNA Nanotubes
6.2.6 DNA Hydrogel
6.3 Dynamic Self-Assembly Systems
6.3.1 Passive Assembly-Disassembly System
6.3.2 Trigger-Induced Autonomous Assembly Systems
6.3.3 Active Assembly Systems
6.3.4 Examples of Dynamic DNA Nanorobots
6.4 Modifications of DNA in Nanostructures
6.5 Nucleic Acid Analogues
6.6 Applications of DNA Nanotechnology
6.7 Summary
References
7: Nucleic Acid in Diagnostics
7.1 Introduction
7.2 Nucleic Acid Techniques in Molecular Diagnostics
7.2.1 Extraction of Nucleic Acids
7.2.1.1 Caesium Chloride/Ethidium Bromide Density Gradient Centrifugation
7.2.1.2 Phenol-Chloroform Extraction
7.2.1.3 Solid-Phase Extraction
7.2.1.4 Applications to Clinical Specimens
7.2.2 Nucleic Acid Amplification Techniques
7.2.2.1 Polymerase Chain Reaction
7.2.2.2 Transcription-Based Amplification Methods
7.2.2.3 Loop-Mediated Amplification Methods
7.2.2.4 Helicase-Dependent Amplification (HDA)
7.2.2.5 Signal-Mediated Amplification of RNA Technology (SMART)
7.2.2.6 Nucleic Acid Signal-Based Amplification (NASBA)
7.2.2.7 Recombinase Polymerase Amplification (RPA)
7.2.2.8 Rolling Circle Amplification (RCA)
7.2.2.9 Strand Displacement Amplification (SDA)
7.3 Nucleic Acid Testing for the Detection of Diseases
7.3.1 HIV-I
7.3.2 Cancer
7.3.3 Prenatal Testing
7.3.4 COVID-19
7.3.5 Infectious Diseases
7.4 Nucleic Acid in Personalized and Precision Medicine
7.5 Conclusion and Future Perspectives
References
8: Nucleic Acid Sensors and Logic Gates
8.1 Brief Overview to Biosensors
8.2 Fundamentals of Nucleic Acid Biosensor
8.2.1 Nucleic Acid Hybridization
8.2.2 Biosensors Based on Nucleic Acid Hybridization
8.3 Basic Design of Nucleic Acid Biosensors
8.3.1 Components of Nucleic Acid Biosensor
8.4 Recognition Elements of Nucleic Acid Biosensor
8.4.1 Aptamers
8.4.2 Riboswitches
8.4.3 DNAzymes
8.5 Types of Readouts in NA Biosensors
8.5.1 Optical Transducers
8.5.1.1 Fluorescent-Based Readout Biosensors
8.5.1.2 SPR Biosensor
8.5.1.3 Nano DLS Biosensor
8.5.2 Electrochemical Nucleic Acid Biosensors
8.5.3 Colorimetric Nucleic Acid Biosensor
8.5.4 Mass-Based Transducers
8.5.5 Piezoelectric Biosensor
8.6 Recent Development of Nucleic Acid Biosensors
8.7 Logic Gates: A Brief Introduction
8.8 Design of Logic Devices
8.8.1 Biomolecular Logic Devices
8.8.2 Nucleic Acid-Based Logic Devices
8.8.3 DNA Tetraplex-Based Logic Devices
8.8.4 Modes of Reuse of DNA Logic Devices for Continuous Operation
8.9 Association of DNA with Other Materials
8.10 Summary
References
9: Nucleic Acid Therapeutics in Cancer Biology
9.1 Introduction
9.2 RNAi Therapeutics
9.2.1 MicroRNA and siRNA
9.2.1.1 miRNA Mimics and antimiRs
9.2.1.2 ASOs
9.2.1.3 AntimiRs
9.2.1.4 Others
9.2.1.5 Delivery System of RNAi Therapeutics
9.2.1.6 Viral Vectors
9.2.1.7 Poly(Lactide-co-glycolide) Particles
Neutral Lipid Emulsions
Neutral Liposome 1,2-Dioleoyl-sn-Glycero-3-Phosphatidylcholine
EnGeneIC Delivery Vehicle Nanocells
Synthetic Polyethylenimine
Dendrimers
Cyclodextrin
Poly(Ethylene Glycol)
Chitosan
N-Acetyl-D-Galactosamine
9.3 Decoy Oligonucleotides
9.4 DNAzyme and RNAzymes
9.5 Conclusion and Future Perspectives
References
10: RNA Vaccines: The Evolution, Applications, and the Challenges Ahead
10.1 History of Vaccine
10.2 Introduction About Vaccination
10.3 Vaccines and Immunization
10.4 Types of Vaccines
10.5 Nucleic Acid (DNA) Vaccines at a Glance
10.6 Nucleic Acid Vaccines
10.7 RNA-Based Vaccines
10.8 Modifications of RNA Vaccines over Time
10.9 RNA Vaccines Against Viruses
10.10 RNA Vaccine and COVID-19
10.11 RNA Vaccine and Cancer
10.12 Limitations and Strategies to Tackle It
10.13 Conclusion
References
11: Nucleic Acid Editing
11.1 Contextual Background: Gene Editing
11.2 Methods in Nucleic Acid Editing: Meganuclease, ZFNs, and TALENs
11.3 CRISPR: From Adaptive Immunity to Genome Editing
11.4 Classification of the CRISPR-Cas Systems
11.5 Structural and Functional Insights of SpCas9
11.6 DNA Base Editing
11.7 RNA Targeting and Single-Base RNA Editing
11.8 Modular CRISPR System
11.8.1 Regulation of Cas9 Protein
11.8.1.1 Evolved or Engineered Cas9 Proteins
11.8.1.2 Transcriptionally Controlled Cas9
Tet System
Cre Dependent System
11.8.1.3 Small Molecule-Controlled CRISPR System
CIP Systems
Intein Splicing System
Systems Based on 4-OHT-ER Mediated Nuclear Translocation
Genetic Code Expansion
11.8.1.4 Light-Controlled Cas9 System
Light-Coupled Split Cas9
11.8.2 Regulation of gRNA
11.8.2.1 Engineering of gRNA
Spacer Length Optimization
Modified RNA
Toehold Switches
11.8.2.2 Small Molecule-Controlled gRNA
11.8.2.3 Light-Controlled gRNA
11.9 Gene Therapy Applications: Somatic Cells
11.10 Germline Editing and Active Genetics
References

Citation preview

Subhrangsu Chatterjee Samit Chattopadhyay   Editors

Nucleic Acid Biology and its Application in Human Diseases

Nucleic Acid Biology and its Application in Human Diseases

Subhrangsu Chatterjee • Samit Chattopadhyay Editors

Nucleic Acid Biology and its Application in Human Diseases

Editors Subhrangsu Chatterjee Department of Biophysics Bose Institute Kolkata, West Bengal, India

Samit Chattopadhyay Department of Biological Sciences Birla Institute of Technology and Science Zuarinagar, Goa, India

ISBN 978-981-19-8520-1 ISBN 978-981-19-8519-5 https://doi.org/10.1007/978-981-19-8520-1

(eBook)

# The Editor(s) (if applicable) and The Author(s), under exclusive licence to Springer Nature Singapore Pte Ltd. 2023 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Preface

Nucleic acids are full of riddles. All our hereditary signatures are coded in the form of long threads of Nucleic Acids, which enable all the living organisms to pass on their genetic information from one generation to the next. After the discovery of DNA double helix in 1953, the structure-function relationship of nucleic acids became the main spotlight in the field of biology and medical science. In the last few decades, we accumulated robust information regarding nucleic acids and their orchestrated functions in both normal and stressed or diseased conditions. Due to enormous technological advancement in the last three decades, the field of nucleic acids research has become tremendously fascinating and a great topic of research worldwide. Now, we have tools to explore more accurate perception regarding different physical and chemical parameters of nucleic acids. Studies on nucleic acids have spread in every sphere globally for a better understanding and cure of several human diseases like cancer, neurodegenerative diseases and infectious diseases. Using Nucleic Acid chain as a template, it has become much easier to detect diseases and the nature of every living organism worldwide. Advancement in the field of biotechnology and bio-molecular science in the last few decades has opened new windows for the development of nucleic acid-based therapeutics for the treatment of several advanced chronic diseases. De novo designed oligonucleotides and their conjugates already have shown enough promises as therapeutic agents for the treatment and diagnosis. Attempts have been made to develop different novel molecules like nanoparticles, oligonucleotide conjugates and selectively modified oligonucleotides thriving enormous impact and possibilities to combat malfunctions in physiology. Thus, nucleic acids have already entered into a diverse research arena. However, even after all this progress, so many questions remain unanswered, leaving a complete void in our understanding. To ponder over the structural dynamics and the role of several canonical and non-canonical nucleic acid structures and their structural intermediates, detailed investigations, both basic and pre-clinical, will be required to unravel all the mysteries. The lack of comprehensive understanding on these integrated issues triggered us to write this book, entitled Nucleic Acid Biology and its Application in Human Diseases.

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Preface

This book is comprised of 11 chapters, written by internationally well-known experts knowledgeable about aspects of nucleic acid structures, biology and therapeutics. This book presents a comprehensive, multidimensional and summarized account of the physicochemical properties of nucleic acids and their role in disease progression with emphasis on their in vivo applications for therapeutic purposes. This book is broadly categorized into five areas: (1) Structure-function relationship of nucleic acids, (2) involvement of nucleic acids in several inflammatory and chronic diseases, (3) nucleic acid-based therapeutic strategies to treat neurodegenerative diseases, cancer, infectious diseases, etc., (4) application of nucleic acid in diagnostics and nanotechnology as sensors and logic gates, (5) frontiers in nucleic acid editing to combat diseases. We discussed recent cutting-edge nucleic acid-based technologies highlighting on the development of antisense oligonucleotides, ribozymes, peptide nucleic acids (PNA) and aptamer nucleic acids for treatment and diagnosis. We tried our best to address the recent advancements and discoveries to enrich the quality of the book, which will surely attract all the aspiring scholars, scientists, researchers from the field of biophysics, biochemistry, molecular biology, disease biology, medicinal chemistry, polymer chemistry, material science, nanotechnology and pharmaceutics across the globe. We wish to thank all the participating authors, whose efforts and top-notch contributions made this book possible. We sincerely thank Prof Klaus Weisz for writing the introductory chapter and setting the standard of this book, which we carried thereafter. We are particularly grateful to all our students for their help, collaboration and never-ending patience during completion of this book. We hope that this book will surely provide the threshold stimulation for the fundamental understanding of nucleic acid structures and their applications in therapeutics to trigger the zeal in aspiring scholars and scientists to cultivate this field to drift it to the next dimension with higher resolution. Cheers to the team effort!!!! Sincerely, Kolkata, West Bengal, India Zuarinagar, Goa, India

Subhrangsu Chatterjee Samit Chattopadhyay

Contents

1

Nucleic Acid Structure and Biology . . . . . . . . . . . . . . . . . . . . . . . . . Yoanes Maria Vianney, Jagannath Jana, Nina Schröder, and Klaus Weisz

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2

Nucleic Acid-Mediated Inflammatory Diseases . . . . . . . . . . . . . . . . Deba Prasad Mandal and Shamee Bhattacharjee

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3

Alternative Splicing and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . Arpankumar Choksi, Richa Pant, Kiran Nakka, Meghna Singh, Akshita Upreti, and Samit Chattopadhyay

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Nucleic Acid-Based Strategies to Treat Neurodegenerative Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 Suman Panda, Oishika Chatterjee, and Subhrangsu Chatterjee

5

Human Diseases Induced by Oxidative Damage in DNA . . . . . . . . . 135 Suman Panda, Oishika Chatterjee, Gopeswar Mukherjee, and Subhrangsu Chatterjee

6

Nucleic Acid in Nanotechnology . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 Debopriya Bose, Laboni Roy, Ananya Roy, and Subhrangsu Chatterjee

7

Nucleic Acid in Diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 Anindya Dutta, Nilanjan Banerjee, Madhurima Chaudhuri, and Subhrangsu Chatterjee

8

Nucleic Acid Sensors and Logic Gates . . . . . . . . . . . . . . . . . . . . . . . 271 Debopriya Bose, Ananya Roy, Laboni Roy, and Subhrangsu Chatterjee

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Contents

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Nucleic Acid Therapeutics in Cancer Biology . . . . . . . . . . . . . . . . . 321 Pallabi Sengupta, Nilanjan Banerjee, Anindya Dutta, Madhurima Chaudhuri, and Subhrangsu Chatterjee

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RNA Vaccines: The Evolution, Applications, and the Challenges Ahead . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349 Ishani Banerji, Shreya Bhattacharjee, Kamalika Mukherjee, and Suvendra N. Bhattacharyya

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Nucleic Acid Editing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365 Ayush Mistry, Sadiya Tanga, and Basudeb Maji

Editors and Contributors

About the Editors Subhrangsu Chatterjee is a faculty member in the Department of Biophysics at Bose Institute, India, since 2011. Prof Chatterjee graduated from the Department of Chemistry, IIT, Kharagpur, in 2002 and later obtained his PhD in Nucleic Acid Structures and Dynamics from the Department of Organic Chemistry and Biochemistry at Uppsala University, Sweden, in 2008. He worked on prion protein misfolding in the Department of Biochemistry at the University of Alberta, Canada, in his postdoctoral studies from 2008 to 2011. His current research is focused on the structure and functions of G-quadruplexes in oncogenic promoters and noncoding RNAs and their implications in transcription and translation modulation as well as Gquadruplex-mediated epigenetic reprogramming to drive cancer progression and metastases. He has co-authored many peer-reviewed publications including Nucleic Acid Research, Journal of the American Chemical Society, Drug Discovery Today, Cell Press, Proceedings of the National Academy of Sciences, ACS and RSC Journals. In his research portfolio, he has first in class anti-metastatic agent M2 for solid tumours and Thermostable and Storage stable Insulin which have generated huge translational values for next generation therapeutics.

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Editors and Contributors

Samit Chattopadhyay is a Senior Professor and Shri B K Birla & Shrimati Sarala Birla Chair Professor in the Department of Biological Sciences at BITS-Pilani, KK Birla Goa Campus. Prof. Chattopadhyay graduated from Calcutta University in 1984 and was awarded PhD from Jadavpur University in 1989. He did his postdoctoral studies in UCONN Health Centre, Farmington, CT, USA, and MIT, Boston, USA. He joined the National Centre for Cell Science, Pune, in 1998 and continued his studies on chromatin remodelling and epigenetics cancer and inflammatory diseases. In the recent past, he was the Director, CSIR-Indian Institute of Chemical Biology, Jadavpur, Kolkata (2014–2019). He is the founder Director of Translation Research Unit of Excellence (TRUE) in CSIR-IICB new campus that started in 2016. He is a fellow of The World Academy of Sciences (TWAS). From 2013, he is serving as the fellow of prestigious Sir J C Bose National Fellowship, DST, India. He is also an elected fellow of all three major science academies in India.

Contributors Nilanjan Banerjee Department of Biophysics, Bose Institute, Kolkata, West Bengal, India Ishani Banerji RNA Biology Research Laboratory, Molecular Genetics Division, CSIR-Indian Institute of Chemical Biology, Kolkata, West Bengal, India Academy of Scientific and Innovative Research (AcSIR), CSIR-Human Resource Development Centre, (CSIR-HRDC) Campus, Ghaziabad, Uttar Pradesh, India Shamee Bhattacharjee Department of Zoology, West Bengal State University, Kolkata, West Bengal, India Shreya Bhattacharjee RNA Biology Research Laboratory, Molecular Genetics Division, CSIR-Indian Institute of Chemical Biology, Kolkata, West Bengal, India Academy of Scientific and Innovative Research (AcSIR), CSIR-Human Resource Development Centre, (CSIR-HRDC) Campus, Ghaziabad, Uttar Pradesh, India Suvendra N. Bhattacharyya RNA Biology Research Laboratory, Molecular Genetics Division, CSIR-Indian Institute of Chemical Biology, Kolkata, West Bengal, India Academy of Scientific and Innovative Research (AcSIR), CSIR-Human Resource Development Centre, (CSIR-HRDC) Campus, Ghaziabad, Uttar Pradesh, India

Editors and Contributors

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Debopriya Bose Department of Biophysics, Bose Institute, Kolkata, West Bengal, India Oishika Chatterjee Department of Biophysics, Bose Institute, Kolkata, West Bengal, India Subhrangsu Chatterjee Department of Biophysics, Bose Institute, Kolkata, West Bengal, India Samit Chattopadhyay Department of Biological Sciences, Birla Institute of Technology and Science, Zuarinagar, Goa, India Madhurima Chaudhuri Department of Biophysics, Bose Institute, Kolkata, West Bengal, India Arpankumar Choksi National Centre for Cell Science, Pune, India Anindya Dutta Department of Biophysics, Bose Institute, Kolkata, West Bengal, India Jagannath Jana Institute of Biochemistry, Universität Greifswald, Greifswald, Germany Basudeb Maji Department of Biology, Ashoka University, Sonipat, Haryana, India Department of Chemistry, Ashoka University, Sonipat, Haryana, India Division of Molecular Medicine, Bose Institute, Kolkata, India Deba Prasad Mandal Department of Zoology, West Bengal State University, Kolkata, West Bengal, India Ayush Mistry Department of Biology, Ashoka University, Sonipat, Haryana, India Gopeswar Mukherjee Barasat Cancer Research and Welfare Centre, Kolkata, West Bengal, India Kamalika Mukherjee RNA Biology Research Laboratory, Molecular Genetics Division, CSIR-Indian Institute of Chemical Biology, Kolkata, West Bengal, India Kiran Nakka Sprott Centre for Stem Cell Research, Ottawa Hospital Research Institute, The Ottawa Hospital, Ottawa, ON, Canada Suman Panda Department of Biophysics, Bose Institute, Kolkata, West Bengal, India Richa Pant National Centre for Cell Science, Pune, India Ananya Roy Department of Biophysics, Bose Institute, Kolkata, West Bengal, India Laboni Roy Department of Biophysics, Bose Institute, Kolkata, West Bengal, India

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Editors and Contributors

Nina Schröder Institute of Biochemistry, Universität Greifswald, Greifswald, Germany Pallabi Sengupta Department of Medical Biochemistry and Biophysics, Umeå Universitet, Umeå, Sweden Meghna Singh Department of Biological Sciences, Birla Institute of Technology and Science, Zuarinagar, Goa, India Sadiya Tanga Department of Chemistry, Ashoka University, Sonipat, Haryana, India Akshita Upreti Department of Biological Sciences, Birla Institute of Technology and Science, Zuarinagar, Goa, India Yoanes Maria Vianney Institute of Biochemistry, Universität Greifswald, Greifswald, Germany Klaus Weisz Institute of Biochemistry, Universität Greifswald, Greifswald, Germany

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Nucleic Acid Structure and Biology Yoanes Maria Vianney, Jagannath Jana, Nina Schröder, and Klaus Weisz

1.1

Introduction

Nucleic acids are central among biomolecules in living organisms, being responsible for storing genetic information and translating this information into the amino acid sequence of proteins. Basic constituents are nucleotides, each composed of phosphoric acid, a pentose sugar, and a nitrogen-containing heterocyclic base. Major nucleobases, attached through N-glycosidic linkages to the sugar moiety, are the purine bases adenine (A) and guanine (G) and the pyrimidine bases cytosine (C), thymine (T), and uracil (U). Whereas deoxyribonucleic acids (DNA) contain 2-deoxyribose, ribonucleic acid structures (RNA) comprise ribose sugars and have the thymine base replaced by uracil (Fig. 1.1). RNA features highly varied and often complex three-dimensional structures to serve its many biological functions. In contrast, DNA is mostly seen as a regular double helix first described by Watson and Crick in 1953 (Watson and Crick 1953). Here, two helical polynucleotides composed of nucleosides that are linked through their 5′- and 3′-hydroxyl groups by a phosphate are wrapped around each other in an antiparallel orientation. With their sugar-phosphate backbone at the periphery of the right-handed helix, base pairs formed between complementary strands stack on each other in the interior of the helix. The specificity of base pairing is responsible for the storage of information and allows for its reliable replication. However, more recent studies have suggested a highly dynamic character of the DNA polymer. In fact, many alternative higher-order DNA structures other than Watson–Crick duplexes have been characterized to date. Such transiently formed non-canonical species are increasingly moving into the limelight, revealing their important regulatory role in a

Y. M. Vianney · J. Jana · N. Schröder · K. Weisz (✉) Institute of Biochemistry, Universität Greifswald, Greifswald, Germany e-mail: [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 S. Chatterjee, S. Chattopadhyay (eds.), Nucleic Acid Biology and its Application in Human Diseases, https://doi.org/10.1007/978-981-19-8520-1_1

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Y. M. Vianney et al.

Fig. 1.1 DNA (left) and RNA segment (right) comprising four nucleotides connected by a phosphodiester linkage. The four different nucleobases are attached to the sugar-phosphate backbone running by convention from the 5′- to the 3′-terminus

variety of cellular processes. These include the regulation of gene expression but also their involvement in other central transactions such as transcription and replication. As a consequence, they have also been associated with mutation sites in cancer cells and DNA damage, representing promising targets for therapeutic interventions. The physiological function of nucleic acids is based on their specific interactions under in vivo conditions and detailed knowledge of their favored structure is a prerequisite for a better understanding of biological processes involving DNA or RNA. Likewise, a rational design of drugs targeting nucleic acids requires structural information of the nucleic acid receptor. In a DNA or RNA polymer, supercoiling and unwinding by helicases may have a major impact on the formation of a particular three-dimensional structure. Also, the specific outer environment with its typical pH, ionic strength, type of cation, molecular crowding, and binding partners like proteins or low-molecular-weight metabolites contribute to the pathway of nucleic acid

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Nucleic Acid Structure and Biology

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folding within the cell. This chapter discusses critical interactions that govern the folding of nucleic acids and outline principles underlying the formation of particular secondary structures. It does not provide a comprehensive survey of the plethora of nucleic acid structural motifs reported to date. Rather, it tries to present some of the major forms that have been of particular interest based on their structural properties and their confirmed or putative biological functions.

1.2

Nucleoside Conformation and Hydrogen Bonding

Nucleic acids are polymers build of phosphate-linked nucleoside units, and favored conformational features of these building blocks are also expected to have a profound influence on the structure of the polymer. Thus, whereas higher-order interactions of the polymer structure may override conformational preferences of the free building blocks, it is in some cases the preferred conformer of the nucleoside that drives folding into a specific DNA or RNA conformation. Consequently, it is necessary to first have a closer look at the properties of the free nucleoside building blocks, their low-energy conformers, and critical interactions favoring one conformer over the other.

1.2.1

Sugar Conformation

The five-membered furanose ring system of ribose or 2′-deoxyribose is puckered with ring atoms twisted out of plane. Such non-planar conformations are best exemplified by cyclopentane with a displaced ring atom traveling in a wave-like fashion all around the ring system virtually without any energy barriers, a process called pseudorotation. Although ring planarity would be energetically favorable based on adopted bond angles of the sp3 hybridized carbons, puckering helps to eliminate unfavorable eclipsed conformations with van der Waals repulsion of the vicinal C-H bonds (Pitzer tension). In contrast to the symmetric all-carbon cyclopentane ring, pseudorotation in a ribofuranose ring system is more restricted and results in various twist and envelope forms of different energy. Although a wide range of different conformations have been observed in nucleic acids, two distinct conformers are generally found to be preferred in free nucleosides but also in polymeric structures, namely C2′-endo and C3′-endo. Here, the C2′ or C3′ atom of ribose is displaced upward out of the plane spanned by C1′-O4′-C4′ and thus located at the same side as C5′ and the base (Fig. 1.2a). Employing the concept of pseudorotation first introduced for cyclopentane, all possible ring conformers can also be expressed by their phase angle of pseudorotation P with 0° < P < 360° and a maximum pucker amplitude νmax (Kilpatrick et al. 1947; Altona and Sundaralingam 1972). Thus, with C2′-endo and C3′-endo characterized by P of 162° and 18°, respectively, these two favored conformers occupy south and north domains of the furanose pseudorotational cycle and are usually designated as S (south) and N (north) conformations.

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Fig. 1.2 (a) Cycle of pseudorotational phase angles with most common C3′-endo and C2′-endo sugar conformations located in the north and south domain, respectively. (b) View onto the sugar ring showing the definition of glycosidic torsion angles χ with anti (left) and syn conformers (right) related through base rotations around the glycosidic bond

In aqueous solution, S- and N-type conformers often exchange in a fast conformational equilibrium with energy barriers of about 20 kJ/mol even when located in polymeric nucleic acids. However, there is a preference for either S or N conformations depending on the nucleic acid secondary structure but in particular on the ribose substituents. Whereas 2′-deoxyribonucleosides slightly prefer a

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C2′-endo or south conformation, ribonucleosides have a strong propensity to adopt a C3′-endo or north conformation. Such preferences result from electron-attracting C2′ substituents such as OH being in an axial position and seem to be governed by superimposed anomeric and gauche effects acting within a nucleoside (Plavec et al. 1993). Sugar conformations of nucleosides being building blocks for nucleic acids can have a profound impact on the formation of DNA or RNA secondary structures. In fact, different morphologies for B- and A-type nucleic acids mainly derive from their different sugar puckers. For N-conformations as in ribonucleosides, C5′ and O3′ are “cis” and give a distance of 5.9 Å between adjacent phosphates in a polynucleotide chain (see Fig. 1.2a). In contrast, with a “trans” arrangement of C5′ and O3′ in S-conformation, corresponding P-P distances amount to about 7.0 Å, giving B-DNA a more extended structure with a pitch (height per turn) of 34 Å compared to only 28 Å in A-DNA with its shorter and broader cylindrical envelope. Consequently, controlling the furanose sugar pucker by introducing appropriate substituents at specific positions has been a frequently used approach in stabilizing or even triggering the formation of a target structure.

1.2.2

Syn/Anti Conformation

The orientation of the nucleobase relative to the sugar moiety in nucleosides is characterized by the glycosidic torsion angle χ defined by atoms O4′-C1′-N9-C4 for purines and by O4′-C1′-N1-C2 for pyrimidine bases. Due to sterically restricted rotational positions, two conformational states, namely syn and anti, can be discriminated (Fig. 1.2b). In the anti conformation, the Watson–Crick face of the nucleobase points away from the sugar, whereas in a syn position the Watson–Crick face and therefore the bulk of a purine ring system are located above the sugar. Generally, the anti conformer with a typical range of glycosidic torsion angles of 180° > χ > -115° for pyrimidines but 180° > χ > -60° for purines is preferred. Thus, in contrast to pyrimidine bases, purines are found to also populate the high anti range between -110° and -60°. There is a strong correlation between north sugar pseudorotamers and anti conformations because the N-type sugar places the nucleobase into a pseudoaxial position that causes unfavorable steric interactions if the base is positioned above the sugar ring in a syn orientation (see Fig. 1.2a). However, quantum-mechanical studies have questioned the general validity of such an interpretation, reporting a syn conformation for free nucleosides being close in energy for south- and north-type sugars (Foloppe et al. 2002). Based on crystal data of S-puckered nucleosides, purine nucleosides only slightly favor anti over syn conformers, whereas pyrimidine nucleosides mostly adopt an anti orientation due to a clash of the pyrimidine 2-oxo substituent with the furanose sugar in a syn conformation (De Leeuw et al. 1980; Sundaralingam 1975). Guanosine seems exceptional in often preferring syn orientations as exemplified by 2′-deoxyguanosine-5′-phosphate that crystallizes in the syn form. Also, equilibria can be shifted toward syn when placing sterically demanding substituents at the C8 and C6 position of purines and pyrimidines, respectively. Thus, 8-bromoguanosine and 6-methyluridine adopt a syn conformation to avoid steric clashes of the bromo

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and methyl substituents with the sugar. Being of biological relevance, oxidative stress may convert 2′-deoxyadenosine dA and in particular 2′-deoxyguanosine dG into 8-oxo-dA or 8-oxo-dG. Apparently, the favorable conversion of the oxidation products from an anti into a syn conformation will change their base pairing properties giving rise to mutations during replication.

1.2.3

Base Pair Arrangements

Natural nucleobases carry several hydrogen bond donor and acceptor sites and may be engaged in the formation of various homo- and hetero-base pairs (Fig. 1.3). Playing a central role in biology, Watson–Crick-type AT or AU base pairs in doublestranded DNA or RNA are stabilized by two hydrogen bonds, whereas three hydrogen bonds are formed in a GC Watson–Crick base pair. Hydrogen bonds in base pairs mutually enforce each other. This cooperativity effect suggests that stable base pairs comprise at least two formed hydrogen bonds. Yet, these hydrogen bonds are weak and with only about 10 kJ/mol mostly electrostatic in nature. Consequently, Watson–Crick hydrogen bonds have only a modest effect on the stability of a double helix, but specific base pairing affords selectivity required for the reliable storage and copying of genetic information. According to a structural modeling, 44 distinct base pairs held together by at least two hydrogen bonds and also including protonated bases can be constructed (Walberer et al. 2003). Requiring two adjacent hydrogen bond donor/acceptor sites complementary to those of its pairing partner, pyrimidine bases C, T, and U can only pair through their Watson–Crick edge, whereas purine bases A and G can also pair through their Hoogsteen edge by recruiting endocyclic N7 and O6/N6 as additional hydrogen bond acceptor/donor (Fig. 1.3a). In case of guanine, pairing may also be possible through N2 and N3 at the sugar edge. AT(U) and GC Watson–Crick base pairs in double helices are isomorph; i.e., the glycosidic bonds of superimposed base pairs are perfectly aligned, resulting in minimal strain upon building a regular duplex. Watson–Crick base pairs play a central role in nucleic acid structures and are basic constituents of genomic DNA. However, the remarkable flexibility in base pairing is exemplified by the observation of stable parallel-stranded helices formed by reverse AT Watson–Crick base pairs (van de Sande et al. 1988). If all nucleotides adopt an anti conformation, formation of Watson–Crick and reverse Hoogsteen base pairs demands an antiparallel strand orientation, whereas reverse Watson–Crick and Hoogsteen paired bases are located in two parallel oriented strands. Hence, an antiparallel duplex with AT Hoogsteen base pairs as observed in a crystal comprises syn-adenosine residues paired with anti-thymidine nucleosides (Abrescia et al. 2002). Non-Watson–Crick base pair arrangements are frequently found in alternative secondary nucleic acid structures and non-helical RNA motifs. Thus, isomorphous AT and GC+ Hoogsteen base pairs, but also homo-purine GG and AA pairs, are constituents of base triplets in triple helices. The CC+ base pair comprises three hydrogen bonds and represents the basic unit in the intercalated tetra-stranded i-motif. Whereas a GU base pair is found in the wobble position of t-RNA, other

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Fig. 1.3 (a) Guanine, cytosine, adenine, and thymine base with atom numbering and hydrogen bond donor and acceptor sites indicated by arrows. Base pairing with at least two hydrogen bonds may involve different edges of a purine base. (b) Exemplary base pairs formed between nucleobases in various nucleic acid structures

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base pair arrangements are often critical contributors to specific DNA and especially RNA folding given only small stability differences between competing conformations.

1.3

Double-Helical Nucleic Acids

1.3.1

A-, B-, and Z-Type Helices

Depending on the type of nucleotides, sequence, relative humidity, and salt concentration, the structure of nucleic acid duplexes can be grouped into three generically different families termed A-, B-, and Z-forms (Fig. 1.4). While A- and B-types adopt a right-handed double-helical conformation with Watson–Crick base pairing and all nucleotides in an anti glycosidic conformation, the Z-form is exceptional in being a left-handed helical structure (Table 1.1). Notably, A-type duplex structures are rather conserved with only a limited spread in their conformational parameters, whereas structures of the B-family show more flexibility with considerable sequencedependent structural variations as represented by additional subtypes such as C-, D-, E-, and T-forms.

Fig. 1.4 Side view of A-, B-, and Z-type double-helical structures. For B-DNA, the location of the major and minor groove is indicated by the filled and unfilled arrow, respectively. Base pairs in the A-form structure show significant inclination and translational x-displacement away from the helix axis. The elongated Z-form helix adopts a left-handed structure with a characteristic zigzag path of its backbone

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Table 1.1 Average structural parameters for A-, B-, and Z-DNA Helix sense Sugar pucker Glycosidic bond Base pairs/turn Twist Rise/base pair Base pair inclination x-Displacement Major groove depth Major groove width Minor groove depth Minor groove width

A-DNA Right-handed C3′-endo Anti 11 32.7° 2.9 Å 12° -4.1 Å 13.5 Å 2.7 Å 2.8 Å 11.0 Å

B-DNA Right-handed C2′-endo Anti 10 36° 3.4 Å 2.4° 0.8 Å 8.5 Å 11.7 Å 7.5 Å 5.7 Å

Z-DNA Left-handed C2′-endoa/C3′-endob Antia/synb 12 -10°c/-50°d 3.7 Å -6° 3.0 Å – – 9Å 4Å

a

For cytidine For guanosine c For CG step d For GC step b

A- and B-type duplexes differ in the sugar pucker of their nucleoside constituents, being largely responsible for the observed morphological differences of the helices. Adopting a C3′-endo sugar conformation, the associated short inter-phosphate distance of 5.9 Å within a polynucleotide strand results in an A-type helix being underwound when compared to a B-type duplex that features sugar moieties with a C2′-endo pucker and inter-phosphate distances of 7.0 Å. As a consequence, doublehelical A-type structures exhibit ~11 base pairs per turn with a corresponding rotation around the z-axis (twist) of 32.7° per base pair. In contrast, overwound B-DNA helices comprise ~10 base pairs per turn with corresponding twist angles of 36°. Whereas the axial rise per base pair in B-DNA amounts to about 3.4 Å and closely follows the van der Waals distance, the axial rise in A-type helices is noticeably smaller and only amounts to ~2.9 Å. Due to an inverse correlation between axial rise and base pair inclination, i.e., the angle between the normal of the base pair plane with the helix axis, the latter derives from a significant positive inclination of ~12° in A-type duplexes when compared to only a very small base pair inclination for B-type helices. A considerable structural departure from A- or B-forms is represented by the lefthanded Z-DNA and Z-RNA duplex, formed primarily by alternating purinepyrimidine sequences like (CG)n but also by some other sequences under appropriate conditions (Hall et al. 1984; Wang et al. 1979). The Z-helix also comprises two antiparallel strands with standard Watson–Crick base pairing but is composed of nucleotides with strictly alternating syn and anti conformations along its backbones. In the representative Z-form of d(CGCGCG), guanosines adopt a syn conformation while pyrimidine nucleosides are restricted to the anti conformation. To maintain Watson–Crick base pairing, local chain reversals occur and generate a zigzag backbone (hence the term Z-form) with a purine-pyrimidine dinucleotide as helical

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repeat. In addition to the different glycosidic torsion angles of purine and pyrimidine nucleotides within the repeat unit and different helical twist angles for purinepyrimidine and pyrimidine-purine steps of about -50° and -10°, respectively, sugars in the dinucleotide are C2′-endo puckered for dC but C3′-endo puckered for dG. Grooves formed in double-helical nucleic acids may not only bind structural water molecules but are important recognition elements for drugs and proteins. The geometry of such grooves critically depends on translational shifts along the x-axis of base pairs with respect to the helix axis and is quantified by their x-displacement. In B-type duplexes, base pairs are in the center of the helix with no significant x-displacement and feature a broad major groove and a narrow minor groove of about equal depth. With a negative x-displacement of approx. -4 Å, base pairs in A-helices are shifted away from the helix axis toward the minor groove, resulting in a deep major groove and a shallow minor groove. In contrast, a positive x-displacement of 3–4 Å shifts base pairs in the Z-helix toward the major groove, making the minor groove very deep whereas the major groove forms a convex surface with part of the bases being exposed to solvent. Genomic DNA within the cellular environment mostly forms B-type duplexes. Transitions into A-DNA observed at low humidity may be attributed to the stabilizing effect of water molecules bound within the B-DNA grooves, in particular within the minor groove with a highly ordered chain of water molecules. In contrast, a greater hydrophobic surface area was proposed for A-DNA. Unlike DNA, RNA duplexes exclusively adopt A-form helices in line with the strong propensity of ribonucleotides to have a north-type sugar pucker. A higher flexibility in the sugar puckering of 2′-deoxyribonucleotides is also reflected in DNA–RNA hybrid duplexes favoring a global A-type structure. Such hybrids are prominent structures in vivo and are formed during transcription of DNA into mRNA or by reverse transcription of viral RNA into its DNA complement. Also, synthetic single-stranded “antisense” oligodeoxynucleotides can be designed to hybridize with a complementary target RNA, e.g., an endogenous mRNA species. As a result, RNA in the formed hybrid is selectively cleaved by the RNase H endonuclease while RNA homoduplexes are kept intact. Formation of the rather unusual Z-type duplex was demonstrated for appropriate DNA as well as RNA oligonucleotides preferably at high salt concentrations. Stabilization by high salt may be attributed to a rather short distance between negatively charged phosphates in opposing strands of the slim and extended Z-helix with high salt decreasing electrostatic repulsion. Originally only a mere curiosity, Z-DNA has attracted interest in biology by demonstrating that genomic right-handed B-DNA can be shifted to the left-handed Z-form by negative supercoiling as induced by processive enzymes such as polymerases and helicases and associated with an underwound helix. This suggested the possibility of localized B-to-Z conformational changes with a transient Z-type helix occasionally induced by physiological processes. Also, certain classes of proteins participating in pathological events were found to bind Z-DNA with high affinity and specificity, indicating a biological role for Z-DNA but also Z-RNA in human disease (Herbert 2019; Rich and Zhang 2003).

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1.3.2

11

Superhelicity and Superhelical Stress

The observation that many DNA species such as viral, bacterial, or plasmid DNA exist in a closed circular form sets the basis of a phenomenon called DNA supercoiling (Vinograd et al. 1965). In contrast to a relaxed linear double-helical DNA, circular DNA but also chromosomal DNA fixed by a protein scaffold can additionally be strained or relieved from torsional stress by winding around itself to form right-handed or left-handed supertwists. The topological state of supercoiled DNA is characterized by three parameters, namely the linking number Lk, the twist Tw, and the writhe Wr. The twist defines the number of helical turns around the duplex axis and the writhe denotes the number of crossovers in the supercoils. The sum of twist and writhe gives the linking number Lk, i.e., the number of times one strand winds around the other strand if the circular DNA is constrained to be in a plane: Lk = T w þ W r

ð1:1Þ

Importantly, Lk cannot be changed without breaking one of the strands, a process enabled by topoisomerases. Also, the majority of DNA has a linking number smaller than a fully relaxed circular DNA as a result of their negative supercoiling (Wr < 0). If superhelical turns in such a closed DNA are removed (Wr = 0), the twist will compensate for these changes to result in an underwound duplex promoting the opening of base pairs when adopting a regular B-DNA conformation. Consequently, negative supercoiling has been demonstrated to assist in structural transitions from B-DNA to Z-DNA or to other non-B DNA structures. B-Z transitions strongly benefit from a negative superhelical stress because the conversion of a single right-handed turn in B-DNA to a left-handed turn in Z-DNA results in a ΔTw = -2 brought about through the relief of negative supercoiling. Also, almost all alternative secondary structures like hairpins, H-DNA, or quadruplexes are underwound relative to double-helical DNA. As a consequence, their formation results in a negative change in twist with partial relaxation of the superhelical stress. It is thus not surprising to find that negative superhelicity may strongly support stabilization of such non-B DNA structures.

1.4

Double-Helical Junctions in Nucleic Acids

Helical junctions are characterized by two or more double-helical segments that intersect at a branch point with axial discontinuities. At the intersection, strands are exchanged between the helical domains with all bases still paired with its Watson– Crick complement or with mismatched or unpaired bases at the branch point (Fig. 1.5). Junctions serve to position helical domains in specific orientations relative to each other and thus determine the global shape of the nucleic acid. Coaxial stacking of two double-helical stems with the formation of a continuous helix is often observed and may provide for significant additional stabilization. Also, due to their high phosphate negative charge density, RNA and DNA junctions may form

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Fig. 1.5 (a) Three-way helical junction in an open conformation with no unpaired bases (left) and a “bulged” junction with additional unpaired bases (right). Coaxial stacking of two helices in the latter conformation is only enabled by unpaired bases in one or more of the three different strands at the junction point. (b) Four-way helical junction. In the absence of metal ions, arms are unstacked and fully extended in a square planar arrangement (left). In the presence of metal ions as observed under physiological conditions, an X-shaped structure with coaxially stacked pairs of arms is formed (right). There is a pair of continuous strands (black) and exchanging strands (red), the latter switching between the two helical stacks at the point of strand exchange. (c) Cruciform formation by a palindromic sequence with a twofold axis of symmetry. Each strand forms a hairpin loop by the disruption of interstrand base pairs in favor of intrastrand base pairs

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specific binding pockets for metal ions. Helical junctions are most common for RNA, but DNA four-way junctions in the form of cruciform structures or as central intermediates in genetic recombination are also of significant biological interest. In general, double-helical junctions have important features that enable them to fold in a characteristic way under physiological conditions while also providing a recognition motif in essential cellular processes. Whereas two-way junctions can be found in self-splicing group I introns, most common types of junctions are the three-way and the four-way junctions, which were found to play important physiological roles (Altona et al. 1996).

1.4.1

Three-Way Junctions

Three-way junctions feature three double-helical arms in which one of the arms partially consists of bases complementary to both of the other two strands (Fig. 1.5a). As a consequence, they are rarely formed from genomic DNA strands with their mutual complementarity but form transiently during DNA replication at the replication fork. On the other hand, three-way junctions represent common structural and functional motifs in various RNA species such as ribosomal RNA. The conformation of the three arms follows the canonical conformation of nucleic acids with their characteristic sugar pucker of the A- or B-form. Topologies of three-arm junctions depend on the presence or absence of unpaired bases at the intersection. A Y-shaped three-way junction in an open unstacked conformation and with similar angles between its three arms A, B, and C is formed for sequences lacking unpaired residues at the point of intersection. Here, coaxial stacking is prevented by the rigidity of the fully paired junction. Such an open conformation of a three-arm junction may also form with unpaired bases under low ionic strength, because electrostatic repulsion between phosphates favors a more extended structure. On the other hand, unpaired bases are associated with less constraints at the junction and in the presence of multivalent cations enable coaxial stacking of two helices with the third helix unstacked and bent away. Depending on the stacked partners, two stacked conformations I and II of different energy, namely A/B and A/C stacking, are conceivable. The energetically favored stacking mode has been shown to depend on the location of the pyrimidine base in the penultimate position preceding the junction in arm A (pyrimidine rule) but is also governed by the stability of the quasihairpin loop at the junction (loop rule) (Wu 2004).

1.4.1.1 Three-Way Junctions in Biology and Technology Early observation of a three-way junction critical for ribozyme activity was reported for the hammerhead motif of viral RNA (Scott et al. 1996). More recently, three-way junctions have often been found in viral genomes such as in HIV-1, where they act as recognition and/or binding sites for proteins important for viral survivability and infectivity (Chu et al. 2019; Song et al. 2021). Three-way junctions were also demonstrated to play critical roles for ribosome function and for telomerase activity (Huang et al. 2014; Réblová et al. 2012).

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Realizing the biological significance of three-arm junctions, attempts have been made to design molecules for their specific targeting. In addition to modulating the cellular formation of three-way junctions, specifically binding ligands can also be used to induce DNA damage by the accumulation of replicative lesions in rapidly dividing cells, e.g., for cancer therapy (Guyon et al. 2018). The triangular cavity in the center of the Y-shaped three-way junction represents a unique structural target for highly specific drugs. Thus, various ligands have been engineered to match the size and shape of this central hydrophobic cavity, optimizing van der Waals interactions but especially face-to-face π-stacking interactions with the three unstacked base pairs at the intersection point. Examples include metallosupramolecular helicates, triptycenes, or carbazole-based macrocycles (Barros and Chenoweth 2014; Oleksi et al. 2006; Yang et al. 2018). In addition to their biological significance, three-way junctions are useful structural motifs for various DNA-based technologies. Thus, three-way junctions with their conformational flexibility have been increasingly exploited as versatile scaffolds for the controlled self-assembly of nanostructures, in the design of optical biomaterials, or in the engineering of nanoswitching devices (Menacher et al. 2011; Seemann et al. 2011; Thomas et al. 2012).

1.4.2

Four-Way Junctions

The first four-way junctions structurally characterized in more detail were cruciform structures formed by inverted repeat sequences. Other four-way junctions comprising four individual strands include the Holliday junction formed during homologous recombination. Four-way junctions are important intermediates in many physiological processes with a variety of biological functions. Like three-way junctions, each double-helical arm of the junction adopts a conformation typical of a B- or A-type nucleic acid.

1.4.2.1 Holliday Junctions The so-called Holliday junction is a four-way junction comprising four strands. It is a key intermediate in site-specific recombination events involving integrases and in homologous recombination, a process that provides for genetic diversity by shifting genes between two chromosomes. Holliday junctions formed during homologous recombination are unique among four-arm junctions in being formed between identical or nearly identical sequences. The symmetric arrangement of sequences around the central junction allows for a branch migration with a crossover of genetic material between the two chromosomes. There are two major forms of four-arm junctions with the favored conformer depending on the ionic strength (Fig. 1.5b). At low-salt concentrations, the Holliday junction exists in a square open-X form; i.e., the four arms extend along different directions and bases are unstacked at the junction. In this extended form, repulsion of the negatively charged phosphate backbone in the center of the junction is minimized. At high salt concentrations, particularly in the presence of multivalent

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metal ions, the four arms arrange into two double-helical domains with continuous coaxial stacking throughout the junction for pairs of helical arms. Here, a specific binding site for metal ions has been reported at the point of strand exchange. While two continuous strands in such a stacked X-shaped structure remain roughly helical and run through the two stacked helices, the two exchanging strands cross from one pair to the other at the crossover point. Two stereochemically equivalent conformers for an X-stacked structure are conceivable and correspond to the two possible choices of stacking partners with different continuous and exchanging strands and relative stabilities depending on local base sequence at the junction (Grainger et al. 1998). While the extended structure is unfavored at higher salt concentrations, it is thought to exist as an intermediate in branch migrations and in crossover isomerizations when going from one conformer to the other (Ortiz-Lombardía et al. 1999).

1.4.2.2 Cruciform Structures Four-way junctions are also adopted by cruciform structures. These may be formed by self-complementary double-helical DNA featuring inverted repeat sequences or palindromes (Fig. 1.5c). In a cruciform species, two B-DNA duplex domains connect with two stem-loop structures with intrastrand base pairing at a four-way junction. A net loss of free energy upon duplex-to-cruciform transitions results from disruption of base pairing in the double-helical structure that is not fully regained through base pair formation within the hairpins comprising unpaired and unstacked bases. However, under conditions of negative supercoiling, superhelical stress can be reduced in a cruciform DNA to make it thermodynamically stable. Also, inverted repeats have to be long enough to overcome the energetic penalty of cruciforms formed from a canonical duplex. This penalty increases with non-perfect inverted repeats, giving rise to potential bulges in the stem-loop domains. In general, the kinetics of duplex-to-cruciform transitions is very slow and associated with high activation energies. This derives from a mechanism that involves base pair opening of the entire inverted repeat followed by a concerted intrastrand base pairing in the stem-loops (C-type pathway). Another so-called S-type pathway has much lower activation energies by only disrupting a few base pairs at a time. Transition to a cruciform structure involves intermediate formation of a proto-cruciform species with short stem-loops followed by elongation of the stems through branch migration. Whereas C-type pathways are favored by sequences rich in AT base pairs up to 100 base pairs away from the inverted repeat and lower salt concentrations to destabilize the double helix, the S-type mechanism is suggested to be less dependent on temperature but sensitive toward the base composition in the center of the palindromic sequence. 1.4.2.3 Four-Way Junctions in Biology and Technology The tertiary structure of four-way Holliday junctions participating in genetic recombination is recognized by various proteins responsible for promoting branch migration and for resolving the junction to again release two independent duplexes. Other physiological processes are also associated with the formation of these alternative

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nucleic acid structures. Thus, Holliday junctions can be formed in human telomeric DNA with complementary 5′-TTAGGG-3′ and 5′-CCCTAA-3′ repeats (Haider et al. 2018). In the absence of telomerase, telomere maintenance can be mediated by recombination enabled by such a Holliday junction intermediate. This lengthening of the telomeres (ALT) pathway helps immortalized cells that lack active telomerase to extend their telomeres through an alternative mechanism. Four-way junctions are also formed to relieve replication stress by stalling replication forks. Thus, stalling can occur in the presence of DNA lesions or another cell machinery responsible for transcription. A replication fork reversal starts with the annealing of the two newly synthesized strands to give a four-way junction structure resembling a Holliday junction and is followed by a restart of the reversed forks. This mechanism enables the original lesion to be either removed before reversed fork restart or bypassed through a template-switching mechanism (Quinet et al. 2017). DNA cruciforms have been shown to be engaged in a wide range of critical biological processes including recombination, replication, and the regulation of gene expression (Brázda et al. 2011). Cruciform-forming sequences can often be found at the origin of replication and in various promoter segments. Thus, while having a positive effect for replication, their deletion from an origin of replication (ORI) on a plasmid was found to be detrimental for the replication process. Owing to their fourway junction, cruciforms like four-stranded Holliday junctions represent unique targets for various DNA binding proteins. Examples include replication proteins, nucleases, transcription factors, helicases, and resolvases. Indeed, an enzyme that resolves a junction is thought to interact with the cruciform structure due to its recognition of the four-arm junction in a similar way as observed for the resolvation of the Holliday junction. Such resolvation of the junction can be used to maintain genomic stability and to recruit further DNA repair mechanisms. Statistical analyses have also suggested a correlation between cruciform but also other non-B DNA structure formation in introns and exon skipping activity (Tsai et al. 2014). Small molecules rationally designed to specifically target four-way junctions can be valuable tools, e.g., as selective inhibitors of Holliday junction resolution or as possible therapeutics interfering with the ALT (alternative lengthening of telomeres) pathway (Brogden et al. 2007). Ligand conjugates were also demonstrated to induce formation of a four-way junction when targeting CTG trinucleotide repeats responsible for neurological diseases such as myotonic dystrophy type 1 (Chien et al. 2020). Here, the core of the formed complex adopts a new type of four-way junction with a U-shaped head-to-head topology and a crossover of all four strands at the junction site. In addition to three-way junctions, four-way junctions also allow the construction of novel nanostructures for various nano- and biotechnological applications. Thus, branched RNA either forming three-way or four-way junctions may be used in RNA interference applications to enhance RNA stability in biological fluids with a concomitant prolonged RNAi effect (Nakashima et al. 2011).

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1.5

17

Pseudoknots

RNA may form tertiary base–base interactions, linking distinct secondary structural motifs. Strictly, tertiary interactions can be defined by interactions between a residue c located between base-paired nucleotides a and b in the primary sequence and another residue d not located between a and b. If a single-stranded region on either side of a hairpin structure folds back to form base pairs with loop nucleotides of the hairpin, a so-called pseudoknot is formed. Initially reported and confirmed experimentally for turnip yellow mosaic virus RNA, pseudoknots are found in all types of cellular and viral RNA (Kolk et al. 1998; Pleij et al. 1985). The simplest pseudoknot is referred to as a hairpin-type (H-type) pseudoknot and comprises two doublehelical stem regions and two single-stranded loops (Fig. 1.6). The two base-paired

Fig. 1.6 (a) Schematic representation of H-type pseudoknot formation with bases of a singlestranded 5′-region pairing with loop nucleotides of the hairpin. (b) Three-dimensional structure of the pseudoknot within gene 32 mRNA from bacteriophage T2 (PDB entry 2TPK). The two helical stem regions are coaxially stacked and single-stranded loop 1 and loop 2 cross major and minor grooves of stem 2 and stem 1, respectively

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stem regions usually coaxially stack on top of each other to form a quasi-continuous helix; however, stacking can be interrupted by intervening nucleotides of a third loop between stem 1 and stem 2. The right-handed A-form helix positions the singlestranded loop 1 toward the major groove of stem 2, whereas loop 2 is located at the minor groove of stem 1. The stability of pseudoknot structures depends on metal ions, salt concentrations, loop length, and nucleotide composition with magnesium ions known to preferentially stabilize pseudoknots over their constituent hairpins.

1.5.1

Pseudoknots in Biology

Pseudoknots can form stable three-dimensional folds and are associated with a diverse range of biological functions. Thus, RNA pseudoknots have been recognized to play important roles in the regulation of protein synthesis and gene expression (Brierley et al. 2008; Peselis and Serganov 2014). Also, the pseudoknotted structure of the human telomerase RNA is required for telomerase activity (Theimer et al. 2005) and pseudoknots are associated with the control of viral RNA translation and the replication of animal and plant viruses, including the coronavirus responsible for severe acute respiratory syndrome (SARS-CoV). A three-stemmed mRNA pseudoknot structure in the coronavirus was found to promote efficient ribosomal frameshifting, while genome analysis revealed its high degree of conservation among coronaviruses, making it a potential target for antiviral therapeutics (Plant et al. 2005).

1.6

Triple-Helical Structures

1.6.1

Intermolecular Triple Helices

Shortly after the discovery of the double helix by Watson and Crick, the existence of triple-helical nucleic acids formed by synthetic polyribonucleotides was reported in 1957 (Felsenfeld et al. 1957). Felsenfeld, Davis, and Rich demonstrated the formation of a three-stranded polynucleotide structure upon the association of polyadenylic acid (polyA) with two strands of polyuridylic acid (polyU). In triplexes, an oligo- or polynucleotide binds to the major groove of a nucleic acid duplex by forming specific Hoogsteen or reverse Hoogsteen hydrogen bonds between nucleobases of the incoming triplex-forming third strand (TFO) and purine bases of the Watson–Crick duplex (Fig. 1.7). Triplexes can be composed of DNA or RNA strands or consist of both in a mixed hybrid. Depending on the 5′-to-3′-orientation of the TFO, two types of triplex structures can be discriminated (for a review see Thuong and Hélène 1993). In a parallel or pyrimidine motif, a homopyrimidine third strand binds parallel to the purine strand of a homopurinehomopyrimidine duplex domain in its major groove to give a pyrimidine-purinepyrimidine (PyPuPy) triplex. A thymine or uracil base of the third DNA or RNA

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Fig. 1.7 (a) Base triplets in a pyrimidine triplex motif with a Hoogsteen hydrogen-bonded third C+ or T (red) to form C+GC or TAT base triplets and in a purine motif with a third reverse Hoogsteen hydrogen-bonded G or A (red) to form GGC and AAT triplets; arrows indicate relative 5′-to-3′-orientations of corresponding strands. (b) Three-dimensional structure of a parallel triplex with a pyrimidine third strand in red color bound in the Watson–Crick duplex major groove (PDB entry 1BWG). (c) Intramolecular H-DNA and *H-DNA formation of a homopurine-homopyrimidine sequence with a mirror repeat. One-half of either the homopyrimidine or homopurine strand folds back to bind into the major groove of the remaining duplex to form a parallel or antiparallel triplex. Additional forms are possible depending on the 5′- or the 3′-half to be single-stranded

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strand is engaged in two Hoogsteen-type hydrogen bonds with the adenine base of the AT base pair, forming a TAT or UAU base triad. Forming two corresponding Hoogsteen hydrogen bonds with a guanine base of a GC Watson–Crick base pair of the duplex, association of a protonated cytosine base C+ of the TFO gives a C+GC triplet. The required protonation at cytosine N3 to form a C+GC triplet with two Hoogsteen hydrogen bonds makes the formation of parallel triplexes dependent on pH. In fact, triplex formation with a C-containing third strand is strongly promoted at acidic pH values below pH 7. Notably, TAT and C+GC triads are isomorphous and allow formation of a regular triple-helical structure without any backbone distortion. In an antiparallel triplex of the purine motif, an all-purine third strand binds in an antiparallel orientation to the duplex homopurine strand in the duplex major groove by reverse Hoogsteen hydrogen bonds. G and A in the TFO recognize GC and AT base pairs to give GGC and AAT triplets. Due to the capability of T in forming both TA Hoogsteen and TA reverse Hoogsteen base pairs, the purine motif also tolerates thymines in its third strand to form TAT base triplets adjacent to GGC and AAT triplets without major structural distortions. For antiparallel triplexes, no protonation is required for the formation of stable base triads, making them relatively insensitive to pH conditions. However, in contrast to a (PyPuPy) triplex, the stability of purinepurine-pyrimidine (PuPuPy) motifs is compromised by base triads not being strictly isomorphous. In line with a parallel and antiparallel TFO alignment upon forming Hoogsteen and reverse Hoogsteen hydrogen bonds, all nucleotides within the triple-helical structure adopt anti conformations. Pyrimidine bases lack another face for bidentate hydrogen bonding with a third incoming base of the TFO; interactions would only involve a single labile hydrogen bond to the 4-amino group of cytosine or the 4-carbonyl function in case of thymine or uracil. Because switching strands of the TFO when binding a duplex would be associated with significant structural distortions of the sugar-phosphate backbone and with the loss of stacking interactions, duplexes forming stable triplexes are limited to homopurinehomopyrimidine sequences. Each pyrimidine interruption within a homopurine strand lowers thermal stabilities, preventing triplex formation with natural bases in vitro for mixed sequences. In addition to sequence and pH, the stability of a triplex structure depends on environmental conditions like cation concentrations and molecular crowding. Whereas cations will screen negatively charged phosphates of the three paired nucleic acid strands, the latter is suggested to stabilize the triplex through hydration effects. The formation of PuPuPy triplexes is often promoted by multivalent cations such as polyamines and cations like Mg2+. In general, however, triplexes form with lower stability than their duplex counterparts.

1.6.2

H-DNA

In addition to the formation of intermolecular triplexes through interactions of a separate single strand with a double-helical homopurine-homopyrimidine target, intramolecular triplex structures can form at double-helical regions with a mirror

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repeat and an arbitrary insert between the mirror-repeated homopurinehomopyrimidine sequences (Fig. 1.7c). A mirror-repeated homopyrimidine tract folds back on itself and pairs within the major groove of the first half of the duplex to form a PyPuPy triplex with the unpaired half of the complementary homopurine strand bent back in an unstructured single-stranded state. This H-form DNA, termed for its H+ dependence or for hinged DNA, is promoted by low pH but also by negative supercoiling. An *H-form of the PuPuPy type results from a fold-back purine sequence aligned antiparallel to the purine strand of the duplex and bound through reverse Hoogsteen hydrogen bonds. Here, no exact mirror symmetry is required due to TAT triads tolerated in addition to AAT base triplets in binding to an AT base pair. Formation of either an H- or *H-triplex depends on pH and the presence of multivalent cations, with the former favoring H-DNA and the latter promoting *H-DNA formation.

1.6.3

Triplexes in Biology and Technology

Bioinformatic analyses have suggested that putative triplex or H-DNA motifs are abundant in the genomes of eukaryotes and prokaryotes where they are found in gene regulatory regions, 3′-untranslated regions, and intron sites which influence regulatory functions in cellular processes. Evidence for their formation in vivo comes from chemical probing, immunofluorescence studies using triplex-specific antibodies, or cellular proteins that specifically interact with triple-helical structures and are found in organisms from bacteria to humans (Van Dyke and Nelson 2013). Intramolecular H-DNA motifs have been linked to the induction of genetic instability (Holder et al. 2015). In addition, DNA triplexes were also shown to be intrinsically mutagenic in mammalian cells, contributing to genetic evolution but also to pathological conditions (Wang and Vasquez 2004). Because triplex-forming motifs tend to be overrepresented in gene regulatory regions, and in particular in promoter regions, triplex formation has been proposed to play significant regulatory roles in vivo. In fact, various studies have suggested triple-helical DNA to be implicated in gene regulation, contributing to transcription, replication, and recombination (Nelson et al. 2012). Current evidence also supports the idea that putative RNA– DNA triplex formation by long noncoding RNAs (lncRNAs) may play critical roles in the regulation of many biological processes (Li et al. 2016). Besides the existence and biological role of triple-stranded structures within cells, intermolecular triplexes have sparked a lot of interest for their use in various technological and medicinal applications. The highly specific recognition of a nucleic acid duplex by a synthetic single-stranded TFO enables targeting of a double-helical sequence without prior denaturation into single strands. Conjugating the TFO with a fluorophore or a chemically active agent, such a triplex technology can be employed for, e.g., genome mapping, site-specific artificial nucleases, gene editing, or the control of gene expression in an anti-gene strategy (Rogers et al. 2005). Major disadvantages include the limited recognition code. Thus, only two out of four Watson–Crick base pairs are recognized by natural bases of the third strand

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associated with the requirement of a homopurine-homopyrimidine sequence. To expand the recognition code and to also allow the targeting of arbitrary mixed double-helical sequences, various synthetic base analogs have been developed that also include cytosine surrogates for pH-independent PyPuPy triplex formation (Hari et al. 2012; Purwanto and Weisz 2003). However, a penalty in triplex stability is generally observed when employing non-canonical base triplets if not strictly isosteric to adjacent canonical triplets.

1.7

The G-Quadruplex

1.7.1

Architecture of the G-Quadruplex Core

G-quadruplexes are four-stranded helical structures that are formed by G-rich DNA and RNA sequences. A guanine tetrad, already reported in 1962 by Gellert et al. when studying the structure of fibers from a gel of concentrated guanylic acid (GMP), constitutes the basic G-quadruplex unit (Gellert et al. 1962). It comprises a square planar arrangement of four guanine bases with fourfold symmetry (Fig. 1.8). The four bases are connected by a cyclic array of eight Hoogsteen hydrogen bonds. The latter involve imino and amino donors at the guanine Watson– Crick side as well as O6 and N7 acceptors at the Hoogsteen side. Hydrogen bonds may progress in a clockwise or anti-clockwise direction if viewed from donor to acceptor sites, resulting in two different tetrad polarities. The helical arrangement of two to four stacked G-tetrads form the G-quadruplex core with three-layered quadruplexes being most common. Stacking is promoted by strong van der Waals interactions of the large tetrad surface areas, but the coordination of monovalent cations within the central cavity of the G-core, in particular potassium or sodium ions, is required for additional stabilization. Due to their smaller size and higher free energy of dehydration, sodium ions show a lower stabilizing effect when compared to larger potassium ions. Notably, folding into quadruplexes is observed under physiological pH and ionic strength conditions and formed quadruplexes may show an even higher thermal stability than duplex DNA (Jana and Weisz 2021). Guanines lined up at each of the four edges of the G-core form a G-column and constitute a tract of ≥2 consecutive guanosine residues in an oligonucleotide sequence. Single G-tracts may be located on four individual strands to form a tetramolecular G-quadruplex. Alternatively, bimolecular or monomolecular G-quadruplex structures result from the folding of two sequences comprising two G-tracts each or of a single sequence comprising four such G-runs, respectively. Small energy differences allow G-residues to either adopt anti or syn glycosidic conformations. Notably, an intact G-tetrad requires all guanine bases of the same tetrad to be oriented the same way. As a consequence, G-residues constituting a tetrad in a G-quadruplex with the same 5′-to-3′ orientation of all four G-columns must adopt the same glycosidic conformation to either form all-anti or all-syn tetrads, the latter being disfavored and only observed in rare cases. On the other

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Fig. 1.8 (a) G-tetrad with a centrally located coordinated metal ion. The direction of hydrogen bonds in either a clockwise or anti-clockwise direction defines the tetrad polarity (indicated by a cyclic arrow). Upon the stacking of ≥2 tetrads to constitute the G-core of a quadruplex, the dimensions of the formed four grooves as well as relative 5′-to-3′ strand orientations of the four G-columns, marked by (+) and (-), are directly related to glycosidic conformations of neighboring residues within a tetrad. (b) Schematic topology, sequence, and three-dimensional structure of an intramolecular G-quadruplex (PDB entry 2LOD) with a propeller loop followed by a diagonal and a lateral loop shown in different colors. There are three syn-G–anti-G–anti-G and one syn-G–syn-G– anti-G columns with two medium (m), one narrow (n), and one wide (w) groove. G-residues adopting syn and anti conformations are colored red and gray, respectively

hand, conservation of the hydrogen-bonded G-tetrad alignment requires residues of the same tetrad but located in antiparallel G-tracts to adopt different glycosidic torsion angles. Whereas relative glycosidic conformations of G-residues in each single G-quartet are fixed by the hydrogen bond alignment between the four guanine bases, the

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pattern of glycosidic bond angles along a G-column is flexible and only restricted by different base stacking energies of neighboring residues. Generally, anti–anti and syn–anti steps are energetically favorable when compared to syn–syn and anti–syn steps. Therefore, the pattern of glycosidic torsion angles along the four G-tracts tends to maximize the number of favored anti–anti and syn–anti steps (Cang et al. 2011). Because bases of the guanosine residues point into different directions when adopting syn and anti conformations, glycosidic conformations of G-residues along a G-tract also determine the relative polarity of stacked tetrads, resulting in homopolar or heteropolar stacking interactions if stacked tetrads feature the same or opposing polarities, respectively. There are four grooves separated by the sugar-phosphate backbone of the four G-columns in quadruplexes. For G-quadruplexes with four parallel G-columns and the same glycosidic conformation for all residues within a G-tetrad, these are of equal medium width. In contrast, mixed syn/anti conformations of tetrad residues in a quadruplex with an antiparallel G-tract alignment will result in different groove geometries also including narrow and wide grooves. Thus, following hydrogen bonds from donor to acceptor, neighboring residues with a syn-G→anti-G hydrogen bond alignment will border a narrow groove but anti-G→syn-G and anti-G→anti-G or syn-G→syn-G alignments will lead to wide and medium grooves, respectively.

1.7.2

G-Quadruplex Topologies

Based on their prominent biological but also technological significance, intramolecular G-quadruplexes formed upon the folding of a single G-rich oligonucleotide have attracted most attention. Thus, G4-forming sequences comprise four Gx-tracts separated by short intervening sequences Ny to give a consensus sequence motif GxNyGxNyGxNyGx with x ≥ 2 and 1 ≤ y ≤ 7 in most cases. Upon folding into quadruplexes, the intervening sequences Ny link the four G-columns of the quadruplex core and may form different types of loops. A propeller (double-chain reversal) loop connects two adjacent parallel strands, a lateral (edge-wise) loop links residues of two adjacent antiparallel strands, and a diagonal loop bridges two non-adjacent antiparallel strands (Fig. 1.8b). Geometric restraints set limits regarding the minimal lengths for the loop-forming sequences. Whereas a single nucleotide provides for a most stable propeller loop in a three-tetrad quadruplex, loops with ≥2 nucleotides are generally required for lateral loops bridging a groove and loops with >3 nucleotides are normally needed for diagonal loops bridging distal edges of a G-tetrad. Unlike standard double-helical DNA, DNA G-quadruplexes may adopt a variety of topologies depending on their loop combinations. A parallel topology has all four G-tracts oriented in the same direction by being connected through three propeller loops. Another major topological family of G-quadruplexes includes (3 + 1) hybridtype structures that comprise three parallel and one antiparallel G-column and may be realized by several loop combinations, e.g., by one propeller loop followed by two lateral loops (hybrid-1). Finally, antiparallel quadruplexes comprise pairs of

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parallel and antiparallel G-columns as formed by three lateral loops or a lateral– diagonal–lateral loop combination. Adding to the remarkable topological variability of G-quadruplexes, propeller and lateral loops may either run in a clockwise or in a counterclockwise direction. In contrast to DNA quadruplexes, RNA quadruplexes feature a limited topological landscape and except for few examples fold into parallel quadruplexes with all G-residues in an anti conformation. The growing number of high-resolution G-quadruplex structures, determined in particular by solution NMR, has also revealed a considerable number of non-canonical G-quadruplex types, further extending the topological landscape beyond regular G-quadruplexes. Thus, G-quadruplexes with broken G-columns involving distinct structural elements like bulges, snapback loops, or V-shaped loops are often observed with significant thermal stability (Jana et al. 2021). Corresponding sequences do not necessarily comply with typical sequences set up for standard structures. Consequently, searching for simple consensus motifs is insufficient for predicting a putative folding into a G-quadruplex; more elaborate considerations will be necessary to indicate G-quadruplex formation of given DNA or RNA sequences.

1.7.3

G-Quadruplexes in Biology and Technology

Experimental findings have firmly established the formation and existence of G-quadruplexes in genomes of eukaryotes, prokaryotes, and viruses (Laguerre et al. 2015; Lam et al. 2013). Biostatistical analyses have suggested about 105 to 106 putative quadruplex-forming sequences in the human genome (Chambers et al. 2015; Todd et al. 2005). These are often found at specific sites with functional significance such as telomeric sequences, promoter regions of oncogenic genes, or 5′- and 3′-untranslated regions (UTR) of mRNA. Strong evidence supports a role for G-quadruplexes in the regulation of many physiological processes but also their involvement in pathological conditions (Hänsel-Hertsch et al. 2017; Varshney et al. 2020). These include, but are not limited to, gene expression, mRNA processing, protein translation, DNA replication, telomere function, maintenance of genome integrity, cancer progression, and degenerative disorders (Rhodes and Lipps 2015). Whereas various proteins comprising transcription factors, helicases, and nucleases were identified to bind quadruplexes with high affinity and also some selectivity, the identification of quadruplex-binding small molecules to stabilize the quadruplex fold has sparked interest in the use of G-quadruplexes as attractive targets for therapeutic interventions (Balasubramanian et al. 2011; Neidle 2017). Thus, novel anti-cancer approaches are based on the stabilization of telomeric quadruplexes through specific binding of ligands, thus preventing telomerase action in cancer cells. Quadruplex-binding ligands can also be employed for binding at G-rich sequences near promoter regions of cancer-related genes like c-myc. Here, stabilized quadruplexes can interfere with RNA polymerase to control corresponding gene expression.

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In addition to their obvious significance in biology and medicine, G-quadruplexes have increasingly been employed for various technological applications (ChiorceaPaquim et al. 2018; Mergny and Sen 2019; Neo et al. 2012). Thus, a G-quadruplex scaffold often constitutes a structural element in nucleic acid aptamers and DNAzymes. Also, newly developed sensor systems for metabolites or metal ions, electronic switches, and nanostructures are often based on the specific properties and structural variability of these tetra-stranded structures.

1.8

The i-Motif

In addition to G-quadruplexes, the i-motif structure represents another tetra-stranded species formed by cytosine-rich sequences. A high-resolution NMR structure of an i-motif (intercalated motif) was first reported by Maurice Guéron in 1993 (Gehring et al. 1993). In this nucleic acid secondary structure, two parallel-stranded CC+ base-paired duplexes associate in an antiparallel orientation. The four-stranded structure has a right-handed helicity with every other hemiprotonated and fully intercalated CC+ base pair provided by one of the two parallel duplexes (Fig. 1.9). As a consequence of the intercalative geometry of consecutive base pairs, there is only poor overlap between the six-membered cytosine bases of the i-motif core that features two wide and two narrow grooves.

Fig. 1.9 (a) Schematic structure of an i-motif in a 3′-E topology with intercalated CC+ base pairs of two parallel-stranded duplexes shown in red and black. (b) Three-dimensional structure of a tetra-stranded RNA i-motif in a 5′-E topology with sequence r(UCCCCC) (PDB entry 1I9K); the same color code as in (a) was used with capping 5′-terminal U residues colored green. The i-motif structure is characterized by two narrow and two wide grooves

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Two competing patterns for intercalation in the i-motif core can be discriminated. The outermost CC+ base pair may either be located at the 5′-end or at the 3′-end, classified as 5′-E or 3′-E topologies, respectively. A major determinant for the preference of either 5′-E or 3′-E conformations derives from sugar–sugar contacts and the extent of C-H1′O4′ hydrogen bond contacts bridging a narrow groove (Leroy et al. 2001). In general, more favorable hydrogen bond interactions promote the formation of the 3′-E conformer. However, these are compromised by a more unfavorable electrostatic repulsion of negatively charged phosphates of the two antiparallel strands across the narrow groove. Nevertheless, 3′-E topologies are generally more stable compared to 5′-E conformations, although relative stabilities also depend on salt concentrations. Thus, electrostatic repulsion of phosphates will increase under low-salt conditions to favor the 5′-E conformer. Equilibria between 5′-E and 3′-E topologies can also be tuned by 5′ overhang sequences contributing to additional interactions (Fig. 1.9b). Due to a required cytosine protonation in forming hemiprotonated CC+ base pairs, the i-motif is most stable under slightly acidic conditions. However, intramolecular i-motifs with four contiguous runs of C-residues have also been found to form at neutral pH. The stability of i-motifs is additionally influenced by the length of the cytosine tracts and the length of intervening sequences that connect the C-tracts to form loops in intramolecular motifs. In general, the stability increases with longer Cn-tracts, but sequences with n > 5 mostly show multiple melting transitions indicating the coexistence of different species. The impact of loop lengths is especially pronounced for the central loop (Cheng et al. 2021). Generally, a long central loop enhances the i-motif stability. Stabilities are also influenced by outer solution conditions. i-Motifs have mostly been investigated under in vitro conditions, typically deviating significantly from the cellular environment with its molecular crowding. Addition of polyethylene glycol (PEG) to mimic molecular crowding effects stabilizes i-motif structures even at physiological pH. The increased stability may be attributed to a higher pKa for cytosine. As a consequence, i-motifs being stable only at low pH under in vitro conditions could be stable at physiological pH in the crowded environment within the cell. Cytosine nucleotides in the i-motif favor a north-type sugar pucker, and northfavoring C-nucleotide analogs are expected to have a stabilizing effect. However, incorporation of a 2-deoxy-2-fluoro-arabinose sugar with a favored south-type sugar pucker stabilizes the i-motif with an increase in cytosine pKa (Assi et al. 2016). Here, the increase in stabilization can be attributed to the fluorine itself, withdrawing electrons and making geminal H2′ of the sugar more electropositive. Upon changing the sugar pucker, the positively polarized H2′ is redirected to be in close vicinity to the phosphate backbone, to O4′ of the sequential sugar, and to the carbonyl of the intercalating cytosine, giving rise to favorable Coulombic interactions.

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i-Motif Structures in Biology and Technology

Staining with antibodies that feature a highly specific binding affinity for i-motif structures but no binding to other DNA structures suggested the existence of i-motifs in nuclei of human cells and in the telomeric region of chromosomes (Zeraati et al. 2018). During the cell cycle, i-motif formation peaks at the G1/S-transition. The formation of G-quadruplexes and i-motifs seems to be highly correlated. By stabilizing i-motifs in the S-phase, characterized by predominant G-quadruplex formation, the amount of i-motifs increases at the expense of G-quadruplex species. Consequently, i-motifs and G-quadruplexes have been proposed to mutually exclude each other and to not coexist (King et al. 2020). Bioinformatic search algorithms found that in analogy to G-quadruplexes i-motifs are predominantly located in gene promoter regions, indicating their regulatory role in gene expression. There are also indications for their influence on replication (Takahashi et al. 2017). Depending on the particular gene, i-motifs can have an up- or downregulating impact on gene transcription. Apart from biological functions exerted by i-motifs, their potential use for technological applications has also been reported. In addition to serving as an element in programmable nanotechnology, their pH-dependent stability makes them highly sensitive and valuable as tunable pH sensors applicable in biological systems. Thus, when combined with a coupled fluorescent dye, the i-motif architecture could be used to measure the intracellular pH (Nesterova and Nesterov 2014). Recently, a structure termed the AC-motif was reported, strongly resembling the i-motif. Here, one of the four cytosine tracts has been exchanged for an adenine tract. This sequence is still able to form an intercalative motif, but instead of intercalating CC+ base pairs, the AC-motif consists of intercalating CC+ and CA+ base pairs. The AC-motif has a better tolerance for higher pH values than the i-motif, but stabilization of this structure requires the presence of mandatory Mg2+ ions. Bioinformatic studies have shown repeats in the human genome able to form this motif, e.g., in the CDKL3 promoter region where it was reported to play a key role in regulating gene expression (Hur et al. 2021).

1.9

Impact of Base Modifications on DNA Structure

When reviewing the various nucleic acid structural motifs with their intrinsic interactions, it becomes apparent that even small modifications of a natural nucleobase may have profound effects on the stability of a particular secondary structure. Substitutions may even select for a specific species among various structures competing under the present conditions. Thus, base modifications can alter pKa values, shifting protonation equilibria in case of protonated bases required for stable hydrogen bonding. Alternatively, capabilities of base functionalities to participate as hydrogen bond donor or acceptor can be modulated or completely suppressed. There is also the possibility of controlling syn–anti equilibria by introducing appropriate substituents. Base-modified nucleotides can be synthetically

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introduced for therapeutic, diagnostic, or technological applications but may also be a result of exogenous mutagens such as alkylating agents or of epigenetic modifications. The impact of the latter on structure and function has received much attention due to its considerable biological significance. A bulky substituent at the C8 position of purine bases will shift syn–anti equilibria in favor of the syn conformer due to unfavorable steric interactions in an anti conformation. Consequently, introducing 8-methyl-guanosine has been shown to promote B–Z transitions and to strongly stabilize the Z-form of DNA in CG repeat sequences with their anti-C–syn-G dinucleotide unit (Xu et al. 2003). Likewise, antiparallel or hybrid-type G-quadruplexes may be selected if an incorporated synfavoring G-analog matches the conformation at the substitution site for the given topology (Haase et al. 2019). On the other hand, oxidative stress generates heightened levels of reactive oxygen species (ROS) that oxidize the genome, with guanine-rich sequences being major targets. Oxidation products like 8-oxo-7,8dihydroguanine located within a G-tract impede the latter from its participation in G-tetrad formation. It has been speculated that an adjoining fifth G-tract often observed in quadruplex-forming promoter sequences of oncogenes acts as a “spare tire” for quadruplex folding, with the damaged G-run extruded into a loop to become a substrate for the base excision repair DNA glycosylases in biologically relevant KCl solutions (Fleming et al. 2015). Triple helix formation with a guanine-rich third strand oligonucleotide may be hampered by physiological potassium ion concentrations due to preferential formation of G-quartets. With a 6-thioguanine 6SG substituted for guanine in the TFO, triplex inhibition through quadruplex formation is mostly eliminated (Olivas and Maher 1995). Based on the square planar arrangement of four guanine bases connected by a cyclic array of Hoogsteen hydrogen bonds and weaker hydrogen bonds involving the 6-thiocarbonyl group as hydrogen bond acceptor, stabilities of a G-quartet but not of a 6SGGC base triplet are expected to be seriously compromised by the 6-thio analog. In addition, coordination of a cation in the central channel of a G-core may suffer from a lower intrinsic affinity of sulfur for K+. Epigenetic modifications may include N7 methylation of guanine bases, again repressing their engagement in G-tetrad formation. One of the major epigenetic processes involves a substitution at the cytosine C5 position with a methyl or other one-carbon groups. Due to a higher pKa of 5-methylcytidine when compared to cytidine, methylation can stabilize those alternative structures that require cytosine protonation. These include triplexes with protonated C+GC base triplets but also i-motif structures with their constituent C+C base pairs. However, altered hydrophobic effects and stacking interactions for the methylated residue may also be major contributors to such stabilizing effects. The epigenetic 5-methylated cytosine is commonly observed at CpG islands and is associated with gene silencing. Because this modification confers increased stability to the i-motif structure, it can impact gene transcription. Interestingly, there is also a difference between cancerous and non-cancerous cells in the methylation pattern of cytosines engaged in i-motif structures (Wright et al. 2020).

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Summary

In contrast to the complex tertiary fold often found for various RNA species, typical DNA is mostly considered to adopt a regular B-type conformation under physiological conditions. It consists of a right-handed double-stranded helix with two antiparallel strands held together by specific Watson–Crick base pairs. Such a structure allows for the reliable storage of genetic information and its transfer to daughter cells upon cell division. However, base pairing can easily be rearranged to form alternative structures that may comprise two, three, or even four strands. Favored conditions for their formation differ and involve alternating purine-pyrimidine sequences for left-handed Z-DNA, inverted repeats for cruciform structures, oligopurine-oligopyrimidine sequences with mirror repeat symmetry for triplehelical H-DNA, and G- or C-tracts for G-quadruplexes and i-motifs, respectively. In most cases, these “perturbed” structures are underwound with respect to predominating B-DNA and strongly promoted by negative supercoiling. Also, a higher negative charge density through their neighboring phosphate groups makes these structures sensitive to the presence of cations like potassium or magnesium ions. More recent findings suggest increasingly complex roles for RNA species within the cell. On the other hand, many of the non-canonical DNA structures are possibly only formed temporarily as a result of protein or metabolite binding or induced by a change of outer conditions. Nonetheless, studies continue to reveal their regulatory functions in a plethora of physiological processes and their participation in various diseases. There is also considerable interest regarding the impact of epigenetic modifications, playing putative roles in the (de)stabilization of biologically active non-B-DNA structures and thus in the regulation of cellular pathways. Based on their specific structural features, alternative nucleic acid structures also constitute excellent targets for exogenous low-molecular-weight agents to be developed for medical treatments. In addition, nano- and biotechnological applications are increasingly based on the diversity and particular properties of the different structures adopted by nucleic acids. Thus, understanding the subtle interplay of steric and electronic effects giving rise to a specific three-dimensional structure will continue to be a primary goal in the structural biology of nucleic acids and be a prerequisite for not only understanding their biological function but also for exploiting their potential in medicinal and technological applications.

References Abrescia NG, Thompson A, Huynh-Dinh T, Subirana JA (2002) Crystal structure of an antiparallel DNA fragment with Hoogsteen base pairing. Proc Natl Acad Sci 99:2806–2811 Altona C, Sundaralingam M (1972) Conformational analysis of the sugar ring in nucleosides and nucleotides. New description using the concept of pseudorotation. J Am Chem Soc 94:8205– 8212 Altona C, Pikkemaat JA, Overmars FJJ (1996) Three-way and four-way junctions in DNA: a conformational viewpoint. Curr Opin Struct Biol 6:305–316

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Nucleic Acid-Mediated Inflammatory Diseases Deba Prasad Mandal and Shamee Bhattacharjee

2.1

Introduction

Nucleic acids are ubiquitously present in all living organisms and have the same basic molecular structure. Therefore, to maintain homeostasis, it is vital for every organism to sense and eliminate non-self or foreign nucleic acids, introduced mainly by viruses and bacteriophages, and altered-self-nucleic acids released from damaged or injured cells. Such nucleic acid immunity supposedly evolved earlier than proteindirected adaptive immunity as the former is found to exist even in ancient life forms such as bacteria and archaea (Hartmann 2017). However, despite the widespread occurrence of nucleic acid immunity in living organisms from bacteria to humans, the armory of defense mechanisms against foreign nucleic acids varies greatly from species to species. All organisms have developed nucleases which cleave those nucleic acids which have been identified as non-self based on their structure, localization, or predominance (Schlee and Hartmann 2016). For example in bacteria, restriction-modification system modifies self-DNA or RNA, thereby facilitating the detection of unmodified foreign or non-self-DNA or RNA which is cleaved by nucleases. In addition, bacteria also have a mechanism to recognize and degrade foreign nucleic acids in a sequence-specific manner using the clustered regularly interspaced short palindromic repeats (CRISPRs)/CRISPR-associated (CRISPR/ Cas) system. Such sequence-specific information is also used by RNA interference (RNAi) mechanisms involving siRNA and miRNA in higher multicellular organisms, especially in invertebrates and plants (Schlee and Hartmann 2016). In vertebrates, the recognition of foreign nucleic acids is further facilitated by certain germline-encoded pattern recognition receptors (PRRs) which can identify

D. P. Mandal · S. Bhattacharjee (✉) Department of Zoology, West Bengal State University, Kolkata, West Bengal, India e-mail: [email protected]; [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 S. Chatterjee, S. Chattopadhyay (eds.), Nucleic Acid Biology and its Application in Human Diseases, https://doi.org/10.1007/978-981-19-8520-1_2

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pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). Such PRRs include a number of highly specialized nucleic acid sensing receptors of the Toll-like receptor (TLR) family (e.g., TLR3, TLR7, TLR8, and TLR9), the RIG-I-like receptor (RLR) family of RNA sensors (e.g., RIG-1, melanoma differentiation associated gene 5), and the cytosolic DNA sensor proteins such as cyclic GMP–AMP (cGAMP) synthase (cGAS), absent in melanoma 2 (AIM2), etc., which can detect pathogen-derived nucleic acids leading to the activation of antiviral and inflammatory response mediated by type I interferons and pro-inflammatory cytokines. In addition to these PRRs, there is another category of nucleic acid receptors which act on foreign nucleic acid directly without inducing the immune system. Such types of receptors include double-stranded RNA (dsRNA)-activated protein kinase R (PKR), adenosine deaminase acting on RNA 1 (ADAR1), and 2′-5′-oligo-adenylate synthetase 1 (OAS1). Recent reports suggest that the two categories of receptors can function in an overlapping manner, such that the cleavage products of the second category of receptors can be targeted by PRRs to induce an immune response (Schlee and Hartmann 2016). Nucleic acid sensors also activate programmed cell death pathways such as apoptosis, pyroptosis, and necroptosis which help in the elimination of the damaged or infected cells and also accentuate the release of inflammatory mediators (Maelfait et al. 2020). Immune reaction to nucleic acids is highly relevant to human diseases. Despite being an indispensable component of anti-microbial defense mechanism and maintenance of homeostasis, erroneous or excessive activation of nucleic acid immune response is injurious and can contribute to pathological inflammation and autoimmunity (Okude et al. 2021). In this chapter, we shall provide a review of the various nucleic acid sensing PRRs, the mechanism of sensing foreign nucleic acids by these receptors, and the downstream signaling pathways activated by PRRs. We shall also focus on the various programmed cell death mechanisms triggered by nucleic acid receptors and their importance in host defense against infection or injury. Finally, we discuss about the inflammatory and autoimmune disorders caused by uncontrolled or erroneous activation of nucleic acid sensors and highlight their emerging importance as therapeutic targets to treat such pathological conditions.

2.2

PRR Receptors

Eukaryotic organisms, ranging from unicellular forms like protozoans to the highest evolved complex metazoans, are equipped with cellular receptors capable of discriminating from self (its own) and non-self (foreign). In the case of unicellular organisms it is all confined in its single cellular entity whereas in multicellular organisms these special receptors were endowed to the phagocytic cells known in various nomenclatures as amebocytes, coelomocytes, hemocytes, or macrophages. Designed to combat with the prokaryotic, fungal, and viral entities, these phagocytic cells have special receptors (both cytoplasmic and membrane bound) to identify the nucleic acid component of the invading microorganisms. Thus, nucleic acids and

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their pattern recognition receptors form a pivotal part of the innate immune system and in immune systems of lesser evolved eukaryotic organisms, where the organism greatly (or rather entirely) depends on its innate wing of defence (Buchmann 2014). PRRs are very broadly divided by the nature of their receptors. The well-accepted classes are (Akira et al. 2006; Kawasaki and Kawai 2014). • • • • • •

TLR: Toll-like receptors NLR: Nucleotide-binding oligomerization domain-like receptors CLR: C-type lectin receptors RLR: RIG-1 like receptors ALRs: Absent in melanoma-2-like receptors cGAS: Cyclic GMP-AMP synthase

As in this chapter we are dealing with nucleic acids activating PRRs, we shall limit our discussion to three of them and exclude C-type lectin receptors as it does not interact with either DNA or RNA.

2.2.1

TLRs

These are highly evolutionarily conserved and one of the best studied PRRs capable of interacting with specific or overlapping PAMPs (Kawasaki and Kawai 2014). As TLRs can be localized both in the cell surface (membrane) and in the intracellular components such as the endoplasmic reticulum, lysosome, or endolysosomes (Kawasaki and Kawai 2014). In fact, the TLRs are broadly classified on the basis of their occurrence viz. cell surface TLRs and intracellular TLRs. The basic structure of a TLR consists of an external leucine-rich repetitive sequence (horseshoe shaped), which is the point of interaction with the PAMPs, followed by a single transmembrane sequence and then finally the endo-domain (cytoplasmic) which hosts the Toll-IL-1 receptor complex and is responsible for the downstream signal transductions (Fig. 2.1). The specific localization of TLRs is extremely crucial for proper signaling outcome. PAMP or DAMP entities interact with the ecto-domain of the TLR in both homo- or hetero-dimeric condition coupled with a co-receptor or accessory molecule. Post-activation the TLRs recruit adaptors like MyD88 or TRIF to its TollIL-1 receptor complex and initiate downward signaling. Usually, TLRs in their ultimate execution lead to the activation of multifaceted kinases such as NF-KappaB, IRF, or MAP which culminates in modulation of chemokines and cytokines including type 1 interferons, all of which are much needed to mount an effective attack on the invading microbes. The ultimate outcome of signaling in terms of the innate response is also much dependent on the type of cell in which signaling is being initiated. The TLRs indicated to react with nucleic acids are TLR3, TLR9, TLR7, TLR8, TLR10 (in Humans), and TLR 13 (in mouse). Of these TLR10 is a cell surface TLR,

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Fig. 2.1 Schematic diagram of TLR (Toll-like receptors) showing the different regions and domains

whereas the rest are located in endosomes and hence are intracellular TLRs (Kawasaki and Kawai 2014).

2.2.1.1 TLR3 TLR3, also referred to as CD283, is a glycoprotein which shares structural and functional similarity and is highly conserved among a variety of species ranging from Drosophila to humans. It is basically related to detection of dsRNA (doublestranded), at least 40–50 bp length, in viral infections and stimulates activation of NF-KappaB and ultimate secretion of type 1 interferons while using TRIF as its only adaptor molecule. TLR3 is largely expressed by dendritic cells in both the placenta and pancreas. 2.2.1.2 TLR9 TLR9, also referred to as CD289, is a protein receptor which is again conserved among animals. It is expressed in more variety of cells as compared to TLR3 and is found in macrophages, dendritic cells, NK cells, and other cells associated with antigen presentation, e.g., dendritic cells, macrophages, NK cells, and other APCs. It is activated by DNA of both bacteria and virus. In a work by Hemmi and co-workers (Hemmi et al. 2000), TLR9 was shown to be activated by unmethylated CpG dinucleotides of bacterial origin.

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2.2.1.3 TLR7 TLR7 is a protein receptor and like the rest of its fellow members in the TLR family is highly conserved. It is responsible for activation with single-stranded RNA virus and is activated by both HIV and HCV (hepatitis C virus). RNA segments rich in guanosine and uridine are targeted by TLR7 in endosomes of B cells and plasmacytoid dendritic cells of the lung, placenta, and spleen. Its gene is situated in the X-chromosome near its fellow TLR8. 2.2.1.4 TLR8 TLR8 like its counterpart the TLR 7 is sensitized by single-stranded RNAs and is expressed in macrophages and neutrophils of lungs. TLR8 ultimately activates IRF5 and results in the secretion of IFNbeta, IL-12p70, and TNF (Moen et al. 2019). Interestingly, it has been reported that some bacteria stimulate membrane-bound TLRs, which abrogates TLR8 signaling (Moen et al. 2019). 2.2.1.5 TLR10 (in Humans) TLR 10 (CD290) is a protein receptor highly expressed in both primary lymphoid chambers like thymus and secondary lymphoid centers like the spleen, tonsils, and lymph nodes. It is found in humans and is a pseudogene in mouse. It is stimulated by dsRNA virus. However, there are reports that it shuts down the immune response (Fore et al. 2020). It is also found in T Reg cells and is controlled by a combined effect of FOXP3 and NF-AT (Fore et al. 2020). 2.2.1.6 TLR 13 (in Mouse) TLR 13 is specifically a murine endosomal TLR not described in humans (Signorino et al. 2014). It is known for its sensitivity for specific bacterial RNA from unmethylated motifs of the large ribosomal subunit (23S rRNA) (Signorino et al. 2014). Though a detailed and well-described role of TLR13 is still wanting, it has been shown that silencing it reduced cytokine induction in DCs during specific bacterial RNA triggers (Hidmark et al. 2012).

2.2.2

NLRs

Nucleotide-binding oligomerization domain (Nod)-like receptors are also referred to as nucleotide-binding leucine-rich repeat receptors or NOD-like receptors (NLRs). Nod-leucine-rich repeats (LRRs), NACHT-LRRs, or CATEPILLER proteins are a group of 23 proteins having a conserved Nod sequence. The intracellular protein receptors that are capable of interaction with both PAMPs and DAMPs are usually internalized during phagocytosis. NLRs structurally are composed of three domains: the middle NACHT domain which is conserved and common for all NLRs and to this portion on C-terminal side is the leucine-rich repeat (LRR) onto which the ligand binds and finally the N-terminal where the caspase recruitment domain (CARD), pyrin domain (PYD), acidic transactivating domain, or baculovirus inhibitor repeats (BIRs) lie (Fig. 2.2).

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Fig. 2.2 Schematic diagram of NLRs (nucleotide-binding oligomerization domain-like receptors) showing structural variations among different types of receptors in the family

The central NATCH acts to oligomerize the other domain which is ATP dependent (Franchi et al. 2006). Of the various subdivisions found in NLRs, Cryopyrin/ PYPAF1/NALP3 is the only one that interacts with nucleic acids. Cryopyrin associates with bacterial RNA and with synthetic purine analogs which indicates this receptor’s affinity toward purine. These receptors regulate Caspase 1 activity by cleaving it with its CARD domain. Cryopyrin forms multi-protein complexes known as inflammasomes which promote quick proteolytic activation of caspase1 from its pro-form. This active caspase 1 initiates the cleavage of pro-form of pro-inflammatory cytokines to its active form, e.g., IL-1β and IL-18 (Franchi et al. 2006).

2.2.3

RLRs

RIG-1-like receptors (RLR), spelt out in full is retinoic acid-inducible gene I, are cytosolic receptors which can detect RNA especially of both host and viral origin. The RLR family has three members, viz.

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Fig. 2.3 Schematic diagram of RLR-RIG-1-like receptors. The structural difference between family members RIG-I, melanoma differentiation-associated protein 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2) has been shown in terms of functional and ligand-binding domains

• RIG-I • Melanoma differentiation-associated protein 5 (MDA5) • Laboratory of genetics and physiology 2 (LGP2) Structurally all RLRs have two basic components for RNA detection, a central helicase along with a terminal carboxyl domain (CTD). Two amino-terminal caspase activation and recruitment domains (CARDs) are found in the case of RIG-I and MDA5 receptors which are otherwise absent in the case of LGP2. In the absence of a ligand (RNA), the structure of RIG-I appears to be a closed structure involving association of specific parts of the helicase and CARD2. In the presence of a ligand, the helicase part encloses the RNA (ligand) tightly between itself and CTD. The wrapping and clamping of the RNA with the helicase and CTD initiates structural changes which exposes the CARD regions for further downward signaling (Fig. 2.3). Though the ligand receptor binding takes place in the receptors’ monomeric forms, further CARD signaling requires oligomeric RGI stabilized by non-degradative K63-polyubiqutin chains. MDA5 receptors rather maintain a more unwrapped configuration in the absence of ligand; however, in the presence of dsRNA (ligand), they assemble in polymeric helical filament-like structures for further CARD signaling (Rehwinkel and Gack 2020). Once oligomerization is done, RLRs interact with mitochondrial antiviralsignaling proteins (MAVS) with their CARD domain. The MAVS after this interacts and activates specific kinases known as TANK-binding kinase 1 (TBK1) and IkappaB kinases-ε (IKK ε). These kinases further activate downstream entities IRF3 and IRF7 which in unison with nuclear factor KappaB enter the nucleus to initiate the transcription of type I interferons along with required antiviral genes to bring about proper immunomodulation to counter the invading viruses (Rehwinkel and Gack 2020). Structurally LPG2 is similar to RIG 1 and MDA5 but is devoid of CARD segments. The helicase unit bears similarity with that of MDA5. Though the

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CTD domain of the LPG2 interacts with viral RNA in a similar fashion to CTDs of RIG1, the CTD of LPG2 has higher affinity to dsRNA than its counterpart found in RIG1. Moreover, LPG2 is more diverse in RNA recognition than other RLRs and is not restricted to the length of RNA or nature of 5′ phosphate ends (Thoresen et al. 2021). LPG2 also interacts with MDA5 and influences its CARD activity in the presence of ligand. The exact nature and functionality of LPG2 is still not clear, but the ATPase activity is important for its interaction with MDA5 (Thoresen et al. 2021). The signaling continues similarly to the other RLRs once MAV activation takes place.

2.2.4

ALRs

ALRs vary in both types and numbers in mammals; for example, humans have four while mice 14 ALR genes. Of these most well-defined ones are AIM2 (absent in melanoma 2) and IFI16 (gamma-interferon-inducible protein Ifi-16 or interferoninducible myeloid differentiation transcriptional activator) (Kumar 2021). AIM2 also known as p210 are members of a family of proteins referred to as p200 or HIN-200 (hematopoietic interferon-inducible proteins with a 200 amino acid repeat). Also known as PYHIN (IFI200/HIN200) protein family (PY—pyrin and HIN—HIN-200 domain containing proteins) these proteins have DNA recognizing domains (Fig. 2.4). MEFV is a gene found mutated in patients suffering from familial Mediterranean fever, which is responsible for inflammasome complex assembly (Kumar 2021). Fig. 2.4 Schematic diagram of ALRs—absent in melanoma-2-like receptors. AIM2 and IFI16 have been shown

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Structurally AIM2 has a pyrin domain (PYD) along with a HIN domain. In the absence of a ligand, PYD and HIN domains interact with each other to form an inert receptor. When dsDNA in the cytosol binds to HIN, the PYD with the association of an adaptor molecule ASC (apoptosis-associated speck-like protein containing a CARD) forms pyrin–pyrin interaction forming an inflammasome recruiting procaspase 1. The conversion of procaspase 1 to active caspase 1 initiates the activation of both IL-1β and IL-18 on one hand and gasdermin D on the other. Thus, pyroptosis and inflammation become the two-horned result of such association. Mitochondrial DNA or nuclear DNA if present in the cytosol acts as a ligand for AIM2 (Kumar 2021). Furthermore, AIM2 activation can have a negative influence on cGAS-STING signaling and the type 1 IFN production (Kumar 2021). IFI16-Is, a PYHIN containing protein often included among ALRs, is an intracellular DNA detecting protein. It associates with STING after simulation and initiates the synthesis and activation of IL-1β (Maelfait et al. 2020).

2.2.5

cGAS

Cyclic GMP-AMP synthase (cGAS) was comparatively recently discovered in 2013 but appears to be the most important of all the cytosolic DNA sensors. Other names include C6orf150, or male abnormal 21 domain containing 1 (MAB21D1) (Fig. 2.5) a 522 amino acid protein encoded by gene is located on human chromosome 6q13 (Yu and Liu 2021). It recognizes DNA in the cytosol irrespective of its sequence (self- or non-self-DNA) but only taking cognizance of the length of the DNA (>20 bp) in question. Thus, even self-oxidized DNA, mitochondrial DNA, viral dsDNA (emerging from reverse transcription), etc., can be sensed by cGAS to further activate the innate response. cGAS-dsDNA complex dimerization is an essential step which is not seen when dsDNA 600 informed genotyped relatives. These mutations resulted in abnormal splicing due to whole or partial exon skipping, intron retentions, destruction, and generation of splicing enhancers and silencers in either exons or introns along with the generation of hypomorphic alleles (Casadei et al. 2019).

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Alternative Splicing and Cancer

3.8.2

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Mutations in Spliceosomal and Trans-Regulatory Splicing Factors

Along with cis-regulatory splice site mutations, trans-regulatory splicing factors also get mutated in many cancers. The majority of these mutations result in altered binding and recognition of splice sites by these factors, which results in aberrant alternative splicing (Bonnal et al. 2020). Frequent somatic mutations in spliceosome proteins and regulatory splicing factors have been correlated with a variety of tumors, including hematological malignancies, breast cancers, pancreatic cancers, mucosal melanomas, uveal melanomas, and lung adenocarcinomas (Bonnal et al. 2020; Yoshida and Ogawa 2014). The most frequently mutated spliceosomal proteins and auxiliary splicing factors associated with cancers are SF3B1, U2AF1, SRSF2, and ZRSR2 (Bonnal et al. 2020; Yoshida and Ogawa 2014). SF3B1 (Splicing factor 3b, subunit 1) recognizes branch-point splice sites and recruits U2 snRNP during alternative splicing. Cancer-associated mutations in SF3B1 have been reported in chronic lymphocytic leukemia (CLL), myelodysplastic syndromes (MDS), primary myelofibrosis, uveal melanoma, vulvovaginal mucosal melanoma, and breast cancer (Fu et al. 2017; Lasho et al. 2012a; Quek et al. 2019; Harbour et al. 2013; Singh et al. 2020; Dalton et al. 2019, 2020; Wang et al. 2016). Predominant mutation in SF3B1 occurs on residue K700 which results in the usage of an alternative branch-point splice site (Wang et al. 2016; Yoshida et al. 2011; Alsafadi et al. 2016; Yin et al. 2019). Other cancer-associated SF3B1 mutations on residues K666, H662, R625, and E622 have also been identified which lead to cryptic splice site recognition (Harbour et al. 2013; Dalton et al. 2020; Yoshida et al. 2011; Alsafadi et al. 2016). Domains of SF3B1 and their frequently mutated sites in various cancers have been represented in Fig. 3.9a. Cancer-associated mutations in SF3B1 result in dysregulated alternative splice forms affecting various biological pathways such as DNA damage response, telomere maintenance, Notch signaling, B-cell signaling, and reprogramming of cellular metabolism (Singh et al. 2020; Dalton et al. 2019; Wang et al. 2016; Yin et al. 2019). U2AF1 (U2 small nuclear RNA auxiliary factor 1) recognizes and binds with the AG dinucleotide of 3′ splice sites and recruits U2AF2 to form a heterodimer complex. Cancer- associated U2AF1 mutations have been reported in MDS, acute myeloid leukemia (AML), myelofibrosis, and lung adenocarcinoma (Bamopoulos et al. 2020; Smith et al. 2019; Graubert et al. 2012; Wang et al. 2019b; Wu et al. 2013; Tefferi et al. 2018a, b; Barraco et al. 2016; Thol et al. 2012; Imielinski et al. 2012). Most common cancer-associated mutations in U2AF1 are located within two zinc-finger domains on residues S34, Q157 (Yoshida et al. 2011; Przychodzen et al. 2013). Other less frequent U2AF1 mutations that are correlated with myeloid malignancies are located on residues A26, R35, R156, and G213 (Przychodzen et al. 2013). Domains of U2AF1 and their frequently mutated sites in various cancers have been represented in Fig. 3.9b. Mutations in U2AF1 lead to recognition of cryptic splice sites flanking the AG dinucleotide of 3′ splice sites resulting in exon inclusion (Nguyen et al. 2018; Fei et al. 2018; Shirai et al. 2015; Ilagan et al. 2014). Mutations in U2AF1 lead to dysregulated alternative splicing affecting various

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Fig. 3.9 Key trans-regulatory proteins and their frequently mutated sites in cancer. (a) SF3B1 contains one UHM domain and eleven HEAT domains. The most frequently mutated residue in SF3B1 is K700 and mutations in this residue are reported to be associated with various hematological malignancies. Other relatively less frequently mutated residues are E622, R625, H662, and K666. (b) U2AF1 contains one RRM domain, one RS domain, and two ZF domains. The most frequently mutated residues in U2AF1 are S34 and Q157. Other relatively less frequently mutated residues are A26, R35, R156, and G213. (c) SRSF2 contains one RRM domain and one RS domain. The most frequently mutated residues in SRSF2 are P95 and P96, which are reported to be associated with various cancers. (d) ZRSR2 contains one RRM domain, one RS domain, and two ZF domains. The most frequently mutated residue in ZRSR2 is R189

pathways such as mitosis, RNA processing, DNA methylation, X chromosome inactivation, DNA damage response, and apoptosis (Przychodzen et al. 2013; Ilagan et al. 2015). SRSF2 (serine/arginine-rich splicing factor 2) is one of the core components of the spliceosome complex. It binds to 5′- and 3′-splice sites and recruits other splicing factors for spliceosome assembly. It interacts with other spliceosomal factors via RS domains and forms a bridge between spliceosome components bound to 5′- and 3′-splice sites such as U1 snRNP and U2AF. Cancer-associated SRSF2 mutations have been reported in AML, MDS, CLL, chronic myelomonocytic leukemia (CMML), myelofibrosis, hepatocellular carcinoma, and breast cancer (Yoshida et al. 2011; Papaemmanuil et al. 2013; Jafari et al. 2018; Luo et al. 2017; Park et al. 2019; Lasho et al. 2012b; Hou et al. 2016; Zhang et al. 2020; Rotunno et al. 2016; Duchmann et al. 2018). Most recurrent SRSF2 mutations have been reported on residue P95 and P96 located within an intervening sequence between the RRM domain and RS domain (Graubert et al. 2012). Domains of SRSF2 and their frequently mutated sites in various cancers have been represented in Fig. 3.9c.

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Gene ontology analysis suggests that SRSF2 mutation ontologies are associated with biological pathways such as ribonucleoprotein complex assembly, mitosis, and DNA damage response (Pellagatti et al. 2018). ZRSR2 (zinc finger CCCH type, RNA-binding motif, and serine/arginine-rich 2) interacts with U2 auxiliary factor heterodimer, which recognizes a functional 3′ splice site in pre-mRNA splicing. In the core spliceosomal mutations, the frequency of ZRSR2 is the least compared to SF3B1, U2AF1, and SRSF2. ZRSR2 mutations are reported in patients with hematological malignancies like AML, MDS, and CMML (Yoshida et al. 2011). These mutations are located across the entire coding region of ZRSR2 and are loss-of-function mutations. These mutations are X-linked and reported predominantly in male patients (Yoshida et al. 2011). Domains of ZRSR2 and their frequently mutated sites in various cancers have been represented in Fig. 3.9d. ZRSR2 mutations result in intron retention, specifically of U12-type introns, while splicing of the U2-type introns is not affected. ZRSR2 mutations lead to aberrant splicing of genes related to cell cycle regulation and MAP kinase signaling pathway (Madan et al. 2015). Other less recurrent splicing factor mutations reported in patients with hematological malignancies are in genes like SF3A1, PRPF40B, U2AF1, SF1, SNRNP200, PRPF8, CSTF2T, DDX1, DDX23, DHX32, HNRNPK, METTL3, PLRG1, POLR2A, PRPF3, RBMX, SRRM2, SRSF6, SUPT5H, TRA2B, U2AF1L4, CELF4, SFRS1, SFRS7, and LUC7L2 (Yoshida and Ogawa 2014; Makishima et al. 2012; Quesada et al. 2012; Ley et al. 2013).

3.8.3

Alteration in Expression of Trans-Regulatory Splicing Factors

Alternative splicing is a very dynamic process involving a great variety of spliceosomal and other regulatory proteins. Mutations in spliceosomal proteins and splicing factors are mostly observed in hematological malignancies. In contrast, solid tumors achieve aberrant alternative splicing due to frequent copy number variation or changes in the expression levels of various splicing factors instead of mutations (Anczukow and Krainer 2016). Two major families of trans-regulatory splicing factors, SR family proteins and hnRNPs, regulate alternative splicing by binding on the cis-regulatory elements (Busch and Hertel 2012). Splicing factors bind to splice sites on pre-mRNA and regulate alternative splicing of their targets in a concentration-dependent manner and dysregulation in the expression of these factors has been associated with various cancers (Geuens et al. 2016; Chen et al. 2012; Long and Caceres 2009). SR protein family is composed of 12 proteins (SRSF1-12), each containing an SR domain and one or two RRM domains. SR domain is important for protein–protein interactions and RRM domains are important for RNA binding. SRSF proteins generally bind to the specific binding sequences on pre-mRNA and promote splicing (Long and Caceres 2009). In contrast to the SR protein family, hnRNPs contain a large variety of RBPs with diverse structural and functional properties. Generally, hnRNPs bind to specific binding elements on pre-mRNA and inhibit the splicing of their targets (Geuens et al. 2016). SR family proteins and hnRNPs were reported to

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Fig. 3.10 Enhanced expression of splicing factors and aberrant alternative splicing in tumor cells. Higher expression of splicing regulators such as SR and hnRNP family proteins promotes aberrant alternative splicing, which contributes to the tumorigenic potential of cancer cells. (Created with BioRender.com)

act as both oncogenes and tumor suppressors. The majority of SR family proteins act as oncogenes while some hnRNPs play an oncogenic role and some act as tumor suppressors (Geuens et al. 2016). Many of the oncogenic SR and hnRNP family proteins get highly expressed in cancer cells due to gene amplification, enhanced transcription, enhanced mRNA, and protein stability. Higher expression of these splicing regulators leads to dysregulation of alternative splicing in cancer cells which further results in enhanced tumorigenesis (Fig. 3.10). SRSF1-6 and SRSF10 have been reported to play an oncogenic role in various cancers affecting a wide variety of cellular functions such as proliferation, apoptosis, senescence, metastasis, cell cycle regulation, migration, cytoskeleton organization, polarity, cell signaling, and metabolism (Dvinge et al. 2016; da Silva et al. 2015; Takeiwa et al. 2020). hnRNP A1, hnRNP A2/B1, hnRNP H, hnRNP M, and hnRNP I (PTBP1) play an oncogenic role in affecting pathways associated with cellular metabolism, apoptosis, proliferation, proteasomal degradation, invasion, and metastasis (Dvinge et al. 2016), whereas hnRNP K has been reported to play tumor suppressor function in hematological malignancies (Gallardo et al. 2015). Apart from SR family proteins and hnRNPs, other splicing factors affect alternative splicing and many of them have been associated with aberrant cancer-associated alternative splicing. These splicing factors generally contain RRM or other RNA-binding domains to interact with pre-mRNA. Few of them have been characterized as potential oncogenes and few as tumor suppressors. The expression pattern of these splicing factors affects splicing events the same as that of SR family proteins or hnRNPs. ESRP1, ESRP2, RBFOX2, PRPF6, QKI, RBM4, RBM5, RBM6, and RMB10 are a few RBPs of other classes of splicing factors that are reported to regulate cancer-associated alternative splicing events.

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Alteration in Pathways Regulating Splicing Factors

Apart from gene amplifications or deletions of splicing factor genes, expression levels and their activity get altered due to aberrant upstream signaling pathways affecting transcriptional, post-transcriptional, and post-translational regulation of splicing factors in a wide variety of cancers (Anczukow and Krainer 2016). Several cancer-associated pathways affect the expression and activity of various splicing factors resulting in dysregulated alternative splicing and further contributing to cancer development and progression (Gonçalves et al. 2018). These pathways include oncogene-driven signaling, growth factors-driven signaling, extracellular matrix-mediated signaling, tumor microenvironment-driven signaling, immune response or inflammation-driven signaling, and stress signaling pathways (Gonçalves et al. 2018). Many cancer-associated signaling pathways such as Wnt/β-catenin, ERK/MAPK, and Notch signaling influence the transcriptional activity of oncogenic facilitators like c-Myc (Dang 2012). Dysregulated signaling pathways leads to overexpression of c-Myc in a wide variety of cancers, which results in higher expression of various splicing factors. Ultimately higher expression of these splicing factors leads to aberrant alternative splicing of their target genes which contributes to cancer progression. c-Myc directly binds to the promoter of splicing factors like SRSF1, hnRNP A1, hnRNP A2/B1, PTBP1, hnRNP H, and Sam68 and promotes their transcription leading to higher expression of these splicing factors (Rauch et al. 2011; Das et al. 2012; Caggiano et al. 2019; David et al. 2010). c-Myc mediated overexpression of splicing repressors hnRNP A1, hnRNP A2/B1, and PTBP1 leads to exclusion of exon 9 and inclusion of exon 10 of PKM gene resulting in higher expression of oncogenic isoform PKM2 (David et al. 2010). c-Myc mediated higher expression of PKM2 is known to promote the Warburg effect in various cancers like gliomas, head and neck cancers, and liver cancers (David et al. 2010; Méndez-Lucas et al. 2017; Gupta et al. 2018). c-Myc induction leads to higher expression of its direct target SRSF1, which promotes aberrant splicing of the MKNK2 and TEAD1 genes. Moreover, SRSF1 cooperates with c-Myc and promotes its oncogenic potential by supporting proliferation and anchorage-independent growth in lung cancer (Das et al. 2012). Another direct transcriptional target of c-Myc is hnRNP H, which regulates A-Raf alternative splicing resulting in inhibition of apoptosis and activation of the ERK pathway (Rauch et al. 2011). Sam68 is another transcriptional target of c-Myc and the expression of Sam68 and c-Myc has a positive co-relation in prostate cancer. c-Myc promotes transcription by binding to its promoter along with increasing the rate of productive splicing of Sam68 by altering the transcriptional elongation rate of RNA Pol II within the gene (Caggiano et al. 2019). A networkbased analysis of colon cancer revealed that ELK1 promotes the expression of c-Myc that regulates alternative splicing of RAC1, NUMB, and PKM via induction of PTBP1, and expression of ELK1 is promoted by the RAS-MAPK pathway (Hollander et al. 2016). Apart from activating the expression of splicing factors, Myc has been reported to regulate the core pre-mRNA splicing machinery by promoting the transcription of the core snRNP particle assembly genes such as

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Fig. 3.11 Signaling pathways regulating the expression of splicing factors via c-Myc. Active Wnt/β-catenin and Notch signaling promote transcriptional activation of the c-Myc gene, whereas active ERK/MAPK pathway leads to activation of c-Myc. c-Myc binds to the promoters of target genes (splicing factors) such as hnRNP A1, hnRNP A2/B1, hnRNP H, PTBP1, SRSF1, and Sam68 and promotes their expression. Enhanced expression of these splicing factors leads to aberrant alternative splicing in cancer cells. (Created with BioRender.com)

PRMT5 and BUD31. This further leads to aberrant pre-mRNA splicing and tumorigenesis (Hsu et al. 2015; Koh et al. 2015). Schematic of signaling pathways leading to increased activation of splicing factors via c-Myc activation has been represented in Fig. 3.11. Apart from c-Myc, other transcriptional factors also play an important role in signaling stimulus-based dysregulation of alternative splicing via regulating the expression of various splicing factors. Wnt signaling is known to be highly activated in colon cancer and promotes the cancer stem cell population. Activation of Wnt signaling is reported to modulate the expression of SRSF3 whose upregulation has been reported in cancer cell proliferation and tumorigenesis in CD133+ colon cancer cells (Corbo et al. 2012). In response to Snail-dependent epithelial to mesenchymal transition (EMT) stimuli, ESRP1 gets repressed. CD44 variable exon skipping is one of the key processes in EMT and ESRP1 is known to inhibit CD44 variable exon skipping. Snail-mediated repression of ESRP1 results in aberrant CD44 variants expression which ultimately promotes EMT (Reinke et al. 2012).

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Apart from transcriptional regulation splicing factors are regulated at the posttranslational level by non-sense mediated decay (NMD). Various reports suggest that ~33% of human alternative splicing contains premature termination codon (PTC) which are subjected to NMD (Lewis et al. 2003; da Costa et al. 2017). The expression of many RNA-binding proteins is subjected to regulation through splicing of their own pre-mRNA. Many SR family proteins contain PTC-containing cassette exons (also known as poison exons) within their mRNAs. The expression level of SR family proteins is autoregulated by NMD which forms a negative feedback loop with their own expression (Lewis et al. 2003; da Costa et al. 2017). In contrast, autoregulation of hnRNPs is subjected to both inclusion and exclusion of PTC-containing sequence (McGlincy et al. 2010; Hase et al. 2006; Ni et al. 2007).

3.9

Role of G-Quadruplex Motif at Splice Sites in the Regulation of Alternative Splicing

G-quadruplex (G4) motifs are the guanine-rich region of DNA that leads to the formation of the non-canonical form of DNA, i.e., non-B-DNA motifs, which have been reported to play a role in the regulation of alternative splicing. Through in-silico study/analyses, scientists have concluded that only half of the exon/intron boundaries decision is made by the splice sites indicating the chance that additional features are also a part of this process including the presence of G4 motifs. As G4s are found to be located near splice sites, it is reported to mediate efficient alternative splicing events in a context-dependent manner (Wu et al. 2022). The region of splicing Quantitative Trait Loci (sQTL) is abundant with G4 motifs and hence gets associated with the exon inclusion during alternative splicing. Moreover, RBPs are also found to be linked with G4s (Georgakopoulos-Soares et al. 2022).

3.9.1

Alteration in Post-translational Modifications, Epigenetic Regulation, and Other Regulatory Mechanisms

Splicing factors are subjected to regulation through various post-translational modifications (PTM) such as phosphorylation, acetylation, ubiquitination, and sumoylation (Katzenberger et al. 2009; Pozzi et al. 2018; Naro and Sette 2013; Nakka et al. 2015; Siam et al. 2019; Choksi et al. 2021). PTMs modulate subcellular localization, activity, and protein turnover of various splicing factors and affect various alternative splicing events resulting in the regulation of various cellular and biological processes. The status of these PTMs is regulated by various cancerassociated pathways and stress response mechanisms which further contribute to the cancer cell proliferation and tumorigenesis (Katzenberger et al. 2009; Naro and Sette 2013; Nakka et al. 2015). Cancer cells are subjected to modulation in epigenetic signatures and changes in epigenetic marks such as histone modifications and DNA methylation on the intragenic regions including exonic and intronic sequences further contribute to

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the cancer-associated alternative splicing. Various chromatin remodeling proteins bind to these altered epigenetic marks and modulate RNA pol II processivity, recruit splicing factors on the newly synthesized pre-mRNA, and regulate the inclusion– exclusion fate of particular exon (Luco et al. 2011; Narayanan et al. 2017). Apart from epigenetic regulation, various cancer-associated alternative splicing events are regulated by various long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) (Urbanski et al. 2018) (Table 3.2).

3.10

Dysregulated Alternative Splicing and Hallmarks of Cancer

In 2000, Douglas Hanahan and Robert A. Weinberg proposed six key hallmarks of cancer which highlight how cancer cells achieve various proliferative advantages over normal cells and acquire unstoppable proliferative and tumorigenic potential (Hanahan and Weinberg 2000). In 2011, Douglas Hanahan and Robert A. Weinberg proposed updated hallmarks of cancer which include previously described six hallmarks and four newly described emerging hallmarks of cancer (Hanahan and Weinberg 2011). The list of all hallmarks of cancer proposed by them includes: (1) Sustaining proliferative signaling, (2) Evading growth suppressors, (3) Activating invasion and metastasis, (4) Enabling replicative immortality, (5) Inducing angiogenesis, (6) Resisting cell death, (7) Avoiding immune destruction, (8) Tumorpromoting inflammation, (9) Genome instability and mutation, (10) Deregulating cellular energetics (Hanahan and Weinberg 2011). The advancement in highthroughput proteomic and genomic techniques and extensive research has revealed that there is coordination between the different hallmarks of cancer to achieve unhampered growth and tumorigenesis. A detailed investigation of various cancer models suggests that dysregulation of alternative splicing actively contributes to these proposed hallmarks of cancer and dysregulation of alternative splicing is proposed as an upcoming hallmark of cancers (Oltean and Bates 2014; Ladomery 2013). The proposed hallmarks of cancer and implicated alternative splicing of important genes are listed in Table 3.3 (Urbanski et al. 2018; Oltean and Bates 2014; Ladomery 2013; Liu and Cheng 2013; Sveen et al. 2016).

3.11

Modulation in Alternative Splicing as a Target for Cancer Detection and Therapeutics

The mammalian transcriptome is comprised of a huge population of pre-mRNAs that undergoes alternative splicing to generate a diversity of mRNAs that are translated into protein isoforms having distinct functions (Scotti and Swanson 2016; Wang et al. 2008). In a highly regulated manner, alternative splicing results in the production of different RNA isoforms from a single gene resulting in proteomic and functional diversity (Urbanski et al. 2018). Because of the complexity of events involved in RNA splicing, the process is susceptible to deleterious mutations and polymorphisms, and any mis-splicing event results in the development of multiple

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Table 3.2 List of alternatively spliced genes and their involvement in various cancers Alternative spliced exon/site/domain Lacks the ligand binding domain encoded by Exons 5 and 6 Exon 9a

Inclusion/ exclusion Exclusion

Inclusion

Acute myeloid leukemia

18 bp insertion at Exons 8 and 9

Inclusion

VCAN (versican) (Sheng et al. 2005) APC (adenomatous polyposis coli) (Neklason et al. 2004)

Chondroitin sulfate beta domain Exon 4

Inclusion

VEGF (Cheung et al. 1998)

Exon 6

Exclusion

AIB1 (Reiter et al. 2001) CCND1 (CyclinD1) (Kim et al. 2009) CASP10 (Caspase10) (Wang et al. 2007) DNMT3B (DNA (cytosine-5-)methyltransferase 3 beta) (Gopalakrishnan et al. 2009) PUF60 (poly-U binding splicing factor 60 kDa) (Matsushita et al. 2006) GLI1 (GLI family zinc finger 1) (Lo et al. 2009) VEGFA (vascular endothelial growth factor A) (Hervé et al. 2005) CD44 (Bánky et al. 2012)

Exon 3 Exon 5

Exclusion Exclusion

Between Exon 5 and 6

Exclusion

Osteosarcoma and prostate cancer Breast and prostate cancer Medulloblastoma and colorectal cancer Non-small cell lung cancer Breast cancer Human bladder cancer Gastric cancer

Exon 5

Exclusion

Colon cancer

Exon 2

Inclusion

Colon cancer

Exon 3 and part of Exon 4 Exon 8a

Exclusion

Glioblastoma multiforme Breast cancer

Exon v2-v10 Exon v3-v10 Exon v6 Exons 19 and 21

Inclusion Inclusion Inclusion Exon skipping

Gene name PPARG (peroxisome proliferatoractivated receptor gamma) (Sabatino et al. 2005) RUNX1 (runt-related transcription factor 1) (Yan et al. 2006) CD99 (CD99 molecule) (Scotlandi et al. 2007)

HER2 (Inoue and Fry 2015)

Exclusion

Exclusion

Type of cancer Colorectal cancer

Breast, urinary bladder cancer Breast cancer

pathologies including cancers (Scotti and Swanson 2016). RNA sequencing has analyzed ~900 somatic exonic single nucleotide variants (SNVs) in cancer patients that disrupt splicing. Defects in alternative splicing may also stem from alterations in regulatory splicing machinery or mutations in splicing regulatory elements of cancer-specific genes (Urbanski et al. 2018). The anomalous splicing isoforms can be exploited as the marker for the prognosis and diagnosis of cancer (Di et al. 2019).

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Table 3.3 List of the hallmarks of cancer and associated alternative splicing of important genes. (All the symbols of hallmarks of cancer have been adapted from the template available on BioRender.com)

Hallmarks of cancer

Associated alternative splicing AR, BRAF, CCND1, EGFR, FGFR2, HER2, HRAS, KRAS, PTEN, RAC1

Hallmarks of cancer

Associated alternative splicing BIRC5, BCL2L1, CASP2, CASP8, CASP9, FAS, CFLAR, MCL1, MDM2, TP53

AIMP2, ANXA7, BIN1, RB1, TP53

CD45, CEACAM1, HLA-G, IL7

CD44, CD82, CDH11, CTTN, ENHA, FGFR2, FAM3B, KLF6, MENA, MST1R, RAC1, RON, TNC

CD44, IRF3, PPARA, RAC1, STAT3

TERT

MLH1, MRE11A, RAC1

FN1, VEGFA

GLS, LDHC, MAX, PFKFB3, PKM

Although there is no drug in the market that modifies alternative splicing, the existing understanding of pathologies related to aberrant splicing events suggests the potential of splicing modulators for novel therapeutics for cancer prevention and

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treatment. Different approaches that are used to achieve modulations in RNA splicing are discussed below.

3.11.1 Use of Antisense Oligos (ASOs) or Splicing-Switching Oligos (SSOs) ASOs are synthetic nucleotide analogs that make use of short nucleic acids to base pair with the targeted sequence through Watson-Crick base pairing. ASOs that typically target splicing are referred to as SSOs. Basically, SSOs are 15–30 mer long ASOs that are designed to base pair with the target sequence and bring about steric hindrance for the splicing factors to bind to pre-mRNA thereby altering the recognition of splice sites by spliceosomes. This results in the modification of normal splicing events and thus prevents the formation of truncated or mutated protein (Havens and Hastings 2016). While the US Food and Drug Administration (FDA) has partially/fully approved the use of ASOs for certain diseases: Vitravene (for CMV retinitis in immunocompromised patients), Kynamro (for familial hypercholesterolemia), Exondys 51 (for Duchenne muscular dystrophy), and Spinraza (for spinal muscular atrophy), the use of ASOs and SSOs for cancer therapy is still under consideration (Stein and Castanotto 2017). Some preclinical studies have shown the promising therapeutic value of antisense oligonucleotides in oncology. STAT3 is a transcription factor that is known to play an important role in activating several oncogenic pathways. To date, it is known to be a difficult druggable target but some reports have suggested the use of antisense technology to target STAT3. AZD9150 (ISIS 481464) is a 16-nucleotide ASO designed to target STAT3 mRNA and downregulate its protein expression. Phase I/Ib trial for intravenous administration of AZ9150 has shown efficacy in a subset of patients with heavily pretreated diffuse large B-cell lymphoma (DLBCL). It has also shown antitumor activity in lymphoma and lung cancer models (Reilley et al. 2018; Hong et al. 2015). KRAS is another example of the most frequently mutated gene in cancer. AZD4785 is a high affinity constrained ethyl-containing ASO, which specifically targets KRAS mRNA resulting in inhibition of downstream effector pathways in cancer cells expressing mutated KRAS. AZD4785 is shown to have antitumor activity in subcutaneous xenograft and patient-derived xenograft (PDX) of KRAS-mutated lung cancer in mice model (Ross et al. 2017). Another promising candidate for cancer therapy is MDM4 as the inclusion of exon 6 during alternative splicing results in its full-length expression in many cancers. In human melanoma cell lines and the PDX mouse model of melanoma, ASO-mediated skipping of exon 6 decreased the abundance of MDM4 and inhibited the oncogenic potential of a wide range of human tumors (Dewaele et al. 2016). Recently a research group identified an antisense oligonucleotide AON-Ex726, which binds to the intronic splicing enhancer (ISE) region of intron 6 of hTERT pre-mRNA and inhibited its telomerase activity and induced apoptosis in glioma cells (Wang et al. 2019c). According to a recent study, SSO-mediated switch in alternative splicing of MNK2 gene (encoding kinase belonging to Ras-MAPK pathway) results in elevation of tumor-suppressive

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Mnk2a isoform in place of pro-oncogenic Mnk2b isoform in glioblastoma cells. The outcome is the activation of the p38-MAPK pathway, re-sensitization of glioblastoma cells to chemotherapy, and reduced glioblastoma growth in vivo (Mogilevsky et al. 2018). The application of SSO has also shown some positive results in modulating the splicing of apoptotic regulator Bcl-x. Administration of Bcl-x SSO with liposome–DNA-polycation nanoparticle-induced the redirection of Bcl-x pre-mRNA splicing from Bcl-xL (anti-apoptotic) to Bcl-xS (pro-apoptotic) in melanoma model resulting in reduced tumor load (Bauman et al. 2010). Recently, a research group designed decoy oligonucleotides specifically targeting splicing factors RBFOX1/2, SRSF1, and PTBT1 to inhibit their splicing and biological activities in conditions where these splicing factors (SFs) are either hyperactive or upregulated. This also resulted in the inhibition of oncogenic properties of different cancer cells expressing these SFs (Denichenko et al. 2019). In addition to the abovelisted cases, many other splice variants are identified (for example, AR-V7 for prostate cancer, ER-α30 for breast cancer, and VDR for colon cancer) which could be targeted for oligonucleotide-based cancer therapy provided their systemic delivery is taken care of (Ciccarese et al. 2016; Zhu et al. 2018; Annalora et al. 2019).

3.11.2 Use of Small Molecules The event of alternative splicing may result in the formation of pro-tumorigenic isoforms, which encourage and accelerate tumor growth, thereby making them excellent therapeutic targets for cancer therapy. While the use of oligonucleotidebased therapies to maintain the splicing isoform ratio in different tumors is gaining attention, the difficulty of delivering these oligonucleotides in specific organs raises a concern. Therefore, efforts are being taken to develop and test small molecule modulators to solve the purpose (Salton and Misteli 2016). FR901464 was identified as the first group of compounds isolated from the fermentation broth of the bacterium Pseudomonas sp. No. 2663 to exhibit anti-tumor properties in mice as well as a human tumor on several xenograft models (Nakajima et al. 1996). Later, it was found that FR901464 and its methylated derivative spliceostatin A (SSA) target SF3b and help in its nuclear retention by inhibiting its pre-mRNA splicing by non-covalently binding to the SF3b sub-complex in the U2 snRNP (Kaida et al. 2007; Corrionero et al. 2011). Similarly, isoginkgetin, a bioflavonoid first identified from Metasequoia glyptostroboides (Dawn redwood), markedly decreased MMP-1 expression in breast carcinomas and melanomas, was later found to have antisplicing activity as it sequesters pre-mRNA in complex A (Yoon et al. 2006; O’Brien et al. 2008). These observations paved the way for small molecules to be utilized as splicing modulators in cancer therapy. Recently an orally available splicing modulator of SF3b complex, H3B-8800, has been identified which competes with pladienolide for binding to SF3b complexes and exhibits killing effects in spliceosome-mutant cancer cells (Seiler et al. 2018). Evading apoptosis is one of the hallmarks of cancer development and upregulation of BCL2 family proteins is frequently observed in many cancer types. So, the therapeutic strategies to

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target anti-apoptotic genes could prove beneficial in cancer therapy. In this regard, a small molecule, E7107, is found to induce selective apoptosis in BCL2A1dependent melanoma cells and MCL1-dependent NSCLC cells (Aird et al. 2019). In another strategy, a series of small molecules including Cpd-1, Cpd-2, and Cpd-3 were used to target kinases like CDK-like kinases (CLKs) and serine-arginine protein kinases (SRPKs) that are crucial for exon selection during alternative splicing. These inhibitors induce splicing changes in genes involved in growth and survival pathways (Araki et al. 2015). Another category of small molecules that stalls spliceosome assembly is the set of inhibitors of HDACs and HATs. These inhibitors were found to block pre-mRNA splicing at intermediate steps of the alternative splicing cycle (Kuhn et al. 2009). An entirely different mechanism by which small molecules show anti-tumor activity is by targeting protein degradation. A series of arylsulfonamides: indisulam, tasisulam, and chloroquinoxaline share a common mechanism of recruitment of RBM39 to CUL4-DCAF15 E3 ubiquitin ligase resulting in polyubiquitination and proteasomal degradation of RBM39. Degradation of RBM39 causes aberrant alternative splicing of pre-mRNA and thus cytotoxicity in various cancer cell lines (Han et al. 2017). A phase II study of indisulam in combination with idarubicin and cytarabine benefitted the heavily pretreated patients of acute myeloid leukemia (AML) with estimated 1-year overall survival of 51% in responders as compared with 8% in non-responders. These studies provided insights into the use of the sulfonamide class of drugs as antineoplastic agents (Assi et al. 2018). Recently a computational approach was used to identify the first-ever small molecule prototype VPC-80051 to specifically target hnRNP A1 RBD. Unlike quercetin, which has inhibitory activities against multiple targets, VPC-80051 shows no off-targets in castrate-resistant 22Rv1 cells. This study provides the basis for future computational studies to develop novel and non-promiscuous small molecules for cancer therapeutics (Carabet et al. 2019).

3.11.3 Use of Monoclonal Antibodies (mAbs) Targeting Deregulated Proteins Identification of transmembrane receptors and extracellular matrix-related proteins arising as a result of alternative splicing in various cancers serves as potential targets for antibody-mediated therapeutic interventions (Tian et al. 2020). In situ delivery of mAb in conjugation with radioactive molecules can ensure its delivery to the target protein in the cancer cells (Bonomi et al. 2013). An extremely well-studied target for mAb-based therapy is epidermal growth factor receptor (EGFR) because of its overexpression in many different types of tumors of epidermal origin like lung, breast, glioma, head & neck, ovarian, and prostate. Since EGFR is also expressed in normal tissues, the use of antibodies like cetuximab and panitumumab may have severe side effects (Ciardiello and Tortora 2008). Therefore, monoclonal Abs targeting tumor-specific EGFR epitopes like EGFR variant III (EGFRvIII) are gaining attention to enhance therapeutic specificity (Klausz et al. 2011). Also, exon 4 deleted EGFR variant (del4 EGFR) found in 10% gliomas, 27% prostate

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cancers, and 33% ovarian cancers is recognized by mAb CH12 where it exhibits anti-tumor activities in vivo (Wang et al. 2012). The alternatively spliced extradomain B (EDB) of fibronectin is the top-described marker of tumor angiogenesis and is strongly expressed in aggressive solid tumors. Out of the three characterized antibodies (F8, B7, and D5) against EDB, F8 recognizes both human and mouse antigens with comparable affinity and represents tumor-targeting abilities (Villa et al. 2008). CD44v6 is an oncogenic splice variant of surface antigen CD44 which is over-expressed in >90% of head and neck squamous cell carcinoma (HNSCC). AbD19384 is an engineered recombinant human bivalent Fab antibody developed to target CD44v6-expressing tumors. Labeling of AbD19384 with 124I serves as a reliable non-invasive imaging technique and also hastens its tumor uptake with target specificity (Haylock et al. 2016). In yet another study, CD44v8-10 was identified as the most promising biomarker for antibody development in the case of advanced gastric cancer (AGC). The results suggested that near-infrared fluorophore- or photosensitizer-conjugated anti-CD44v9 antibody could be used for targeted imaging and photoimmunotherapy of gastric cancer (Choi et al. 2017).

3.11.4 Use of Chemotherapeutic Drugs to Modify Splicing Chemotherapy is the most commonly used routine for cancer treatment and is considered the gold standard to date (Lambert et al. 2017). There are several numbers of evidence by different groups which suggest that large numbers of AS events are regulated by genotoxic stress inducers like chemotherapeutic drugs and radiations (Lambert et al. 2017; Dutertre et al. 2011). Camptothecin (CPT) and its known clinical derivatives, topotecan, and irinotecan, are extensively used in cancer therapeutics. They are known to target topoisomerase I (Topo I) which is coupled with splicing by regulating SR-rich splicing proteins. The ExonHit Human Splice Array was used to identify the splice variants with high sensitivity and precision wherein it was found that CPT treatment specifically affects the splicing of RBM8A through Pol II hyperphosphorylation (Solier et al. 2010). A recent study identified that cisplatin (a platinum-based compound) provoked changes in 717 splicing events, many of which were involved in cell cycle regulation. Cisplatin-induced reprogramming of AS is caused by SRSF4 and requires class I PI3Ks P11oβ for inducing apoptosis. Moreover, knock-down of SRSF4 resulted in abrogation of cisplatin-induced changes in splicing events making it an important factor required for anti-cancer properties of cisplatin (Gabriel et al. 2015). Paclitaxel, a first-line chemotherapeutic drug used for the treatment of advanced non-small-cell lung carcinoma (NSCLC), modulates the splicing of ECT2 (an important factor implicated in the regulation of cytokinesis). Paclitaxel can inhibit cancer cell proliferation by switching the full-length ECT2 to its short splicing isoform ECT2-S1820.

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3.11.5 Use of Spliceosome-Mediated RNA Trans-Splicing (SMaRT) Technique Trans-splicing is the process in which exons from different transcripts are joined together to form a new transcript. It is used as a therapeutic approach for the modulation of alternative splicing of genes responsible for the disease progression. It includes two approaches, i.e., (1) Ribozyme mediated and (2) SMaRT based. In the first method, an artificial ribozyme is synthesized to bind and trans-splice the targeted RNA sequence. The limitation in this method is the presence of secondary structures in the ribozyme, which makes it unable to bind to the target sequence sometimes. Also, the reaction mediated by the ribozyme is reversible and it takes a high cost to synthesize the ribozyme, whereas SMaRT is a therapeutic process in which endogenous spliceosome machinery is used and artificial RNA trans-splicing molecules (RTMs) are given to the cells. RTMs splice out the mutated region of the target RNA and replace it with the inclusion of respective wild-type coding regions. For the trans-splicing, RTM must possess (1) wild-type coding region that needs to exchange, (2) target recognition sequence, (3) spliceosome binding site, and (4) splice sites. Trans-splicing is done by replacing exons in three ways such as 5′ exon splicing, 3′ exon splicing, and internal exon splicing. SMaRT has been proved to efficiently work as a therapeutic for target genes both by in vivo and in vitro studies. In animal models, RTMs containing a 3′ splice site replaces the mutated genes like survival motor neuron (SMN2), collagen VII, CD40L, and factor VIII (F8) for the treatment of spinal muscular atrophy, dystrophic epidermolysis bullosa, hyper-IgM X-linked immunodeficiency and hemophilia A, respectively (Chao et al. 2003; Tahara et al. 2004; Mayr et al. 2022; Shababi et al. 2011). Also, it is being applied to in-vitro studies to correct genes such as cystic fibrosis transmembrane conductance regulator (CFTR), β-globin, survival motor neuron 2 (SMN2) in disease cystic fibrosis, sickle cell anemia, spinal muscular atrophy respectively also found to correct some genes in other diseases (Kierlin-Duncan and Sullenger 2007; Liu et al. 2005; Coady et al. 2008). The limitation of the SMaRT technique involves the possible homology in the sequence of RTM binding region and the location of the 5′ or 3′ splice site (Wally et al. 2012). In addition to all these methods, clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) systems can be used to manipulate RNAs in living cells (Pickar-Oliver and Gersbach 2019). In a recent study, a CRISPR/Cas9 screen was performed to identify numerous dysregulated RNA-binding proteins (RBPs) critical for acute myeloid leukemia (AML) maintenance. CRISPR-mediated deletion of RBM39 resulted in altered splicing of HOXA9 target genes that are required in AML (Wang et al. 2019c). Moreover, RNA-seq holds enormous potential for quantitatively studying alternative splicing in cancer. The use of RNA-Seq requires intense knowledge of statistical and computational tools to augment the diagnosis rate by 10–35% (Marco-Puche et al. 2019). The development of bioinformatics tools and integrating them with omics data can lead to a better understanding of cancer development and metastasis (Feng et al. 2013).

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Nucleic Acid-Based Strategies to Treat Neurodegenerative Diseases Suman Panda, Oishika Chatterjee, and Subhrangsu Chatterjee

4.1

Introduction

Due to the technological advancements in the last few decades, we identified and characterized several small RNAs, lncRNAs. Several researches have shown the regulatory role of these RNAs in gene expression leading toward the understanding of structure–function relations and networking of these RNAs to maintain the cellular equilibrium. Chemically modified antisense oligonucleotides can specifically bind to RNA transcribed from the target genes through complementary base pairing and alter post-transcriptional modification and translation. These small RNAs and lncRNAs can alter gene expression in time- and tissue-dependent manner, thus making them a new potential therapeutic target. Due to tissue selective delivery, availability of low-cost delivery systems, and long-term therapeutic effects than conventional protein targeted drugs, nucleic acid-based therapeutics are emerging as a reliable therapeutic window in the treatment of various human diseases. At the dawn of this concept, both siRNA and antisense compounds were used clinically to tune the gene expression of hepatocyte cells. In normal scenario, oligonucleotides do not pass through the healthy blood–brain barrier. But practicable localization of oligonucleotides in brain and spinal cord is achieved through the direct administration of oligonucleotides in the brain parenchyma or cerebrospinal fluid (CSF). But the recent acclamation of Nusinersen in 2016, marketed as Spinraza, a spliceswitching antisense oligonucleotide (ASO) for the treatment of spinal muscular atrophy (SMA), a rare neuromuscular disorder, paved the way toward a new horizon in nucleic acid-based therapeutics for the treatment of neurodegenerative diseases (Hirunagi et al. 2022). After this first approval, several other antisense oligonucleotides got the permission for clinical trials in nucleic acid-based therapies

S. Panda · O. Chatterjee · S. Chatterjee (✉) Department of Biophysics, Bose Institute, Kolkata, West Bengal, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 S. Chatterjee, S. Chattopadhyay (eds.), Nucleic Acid Biology and its Application in Human Diseases, https://doi.org/10.1007/978-981-19-8520-1_4

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and adeno-associated virus vectors (AAV) expressed gene therapies against diseases like MAPT (Tau)-driven Alzheimer’s disease, Huntington’s disease (HD), SOD1and C9ORF72-driven amyotrophic lateral sclerosis (ALS), etc. (Evers et al. 2015) (Table 4.1). Thus, in recent years, treatment of genetically well-defined neurodegenerative diseases has emerged as one of the most reliable segments for nucleic acidbased therapeutics. In this chapter, we will review all the important platform technologies available for neurotherapeutics for the treatment of several neurodegenerative diseases like Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, etc. (Fig. 4.1). We also highlight the opportunities and challenges of these clinically relevant therapies. In addition, we will focus on the development of cutting edge as tissue-specific messenger RNAs, siRNAs, nucleic acid bioconjugates, and gene-editing tools.

4.2

Technologies Under Nucleic Acid Therapeutics

4.2.1

Antisense Oligonucleotides (ASO)

Antisense oligonucleotides (ASOs) are short, single-stranded, synthetic nucleic acids about 8–30 nucleotides long that can hybridize with target cellular RNA through canonical Watson–Crick base pairing to modulate gene expression (Lundin et al. 2015). After binding with the target mRNA or pre-mRNA, ASOs can alter protein synthesis from that target gene through altering post-transcriptional modification, pre-mRNA processing and splicing, providing steric hindrance to translational machinery and degradation of bound target RNA mediated by RNase H1 and Argonaute 2 in RNA interference pathway. The specific interaction between ASO and target RNA also limits the off-target effects. Due to this specificity, 100% targeting of single-nucleotide polymorphism in HTT gene (codes for huntingtin protein) has been achieved through ASOs. Selective design, off-target effects, and molecular size give ASOs a huge advantage and potential therapeutic agent than other nucleic acid-based drugs. The specific binding between ASO-RNA is highly regulated by the complementarity between the full-length ASO molecule and the target RNA. As the length of the ASOs is in the range of ~30 nucleotides, it can inflame cellular toxicity through off-target binding. During administration of these ASOs in vivo, unmodified phosphodiester ASOs are degraded rapidly by the action of several endo- and exo-nucleases present in the serum and excreted by renal filtration, thus decreasing the half-life and efficacy of ASOs. To improve the pharmacokinetics, pharmacodynamics, and efficacy in binding their mRNA target, the heterocyclic nucleobase, the ribose sugar moiety, the phosphodiester linkage, or the sugar-phosphate backbone of ASOs has been chemically modified (Deleavey and Damha 2012). Heavily modified and functionalized ASOs do not even require carrier for delivery thus limiting downstream processing and decreasing production cost. In case of morpholino ASOs, thiophosphoramidate, and PS ASOs, the phosphodiester bond along the backbone has been chemically

Boston Children’s hospital

AveXis/ Novartis

Ionis pharmaceuticals/ Roche Ionis pharmaceuticals/ Biogen Alnylam Pharmaceuticals

–

Onpattro

Zolgensma

–

BIIB067

ALNTTRsc02

Milasen

Patisiran

Onasemnogene abeparvovec

Tominersen

Tofersen

Vutrisiran

Alnylam Pharmaceuticals

Ionis pharmaceuticals

Tegsedi

Inotersen

Company/ organization Biogen

Market name Spinraza

Drug Nusinersen

Superoxide dismutase 1 (SOD1) Transthyretin (TTR)

ASOs

GalNAc–si RNA conjugates

Survival of motor neuron 1 (SMN1) Huntingtin (HTT)

Major facilitator superfamily domain containing 8 (MFSD8) Transthyretin (TTR)

Gene target Survival of motor neuron 2 (SMN2) Transthyretin (TTR)

AAV vectors mediated gene therapy ASOs

LNP-siRNA

ASOs

ASOs

Chemical platform ASOs

Hereditary transthyretin amyloidosis

Amyotrophic lateral sclerosis

Hereditary transthyretin amyloidosis Spinal muscular atrophy Huntington’s disease

Indication Spinal muscular atrophy Hereditary transthyretin amyloidosis Batten disease

Table 4.1 Present clinical status of various nucleic acid-based therapeutics against neurodegenerative disorders

Subcutaneous

Intrathecal

Nucleic Acid-Based Strategies to Treat Neurodegenerative Diseases (continued)

Phase 3 clinical trial NCT03 761849 Phase 3 clinical trial NCT02 623699 Phase 3 clinical trial NCT03 759379

Approved in 2019

Intravenous

Intrathecal

Approved in 2018

Approved in 2018

Approved in 2018

Present status Approved in 2016

Intravenous

Intrathecal

Subcutaneous

Administration route Intrathecal

4 107

AVXS-101

Drug AKCEA-TTRLRx

Company/ organization Akcea/Ionis pharmaceuticals

Novartis

Market name –

–

Table 4.1 (continued)

AAV vectors mediated gene therapy (AAV9)

Chemical platform GalNAc-A SO conjugates Survival of motor neuron 2 (SMN2)

Gene target Transthyretin (TTR)

Indication TTR-mediated amyloid polyneuropathy Spinal muscular atrophy Intrathecal

Administration route Subcutaneous Present status Phase 3 clinical trial NCT04 136184, NCT04 136171 Phase 3 clinical trial NCT03505099, NCT03461289, NCT03306277, NCT03837184

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Fig. 4.1 Modes of action for all the approved in vivo nucleic acid therapeutics; ASO (antisense oligonucleotides) can alter protein synthesis from that target gene through altering posttranscriptional modification, pre-mRNA processing and splicing, providing steric hindrance to translational machinery and degradation of bound target RNA; GalNAc–siRNA conjugates platform is an efficient way to increase the cellular uptake and target organ accumulation siRNA; lipid nanoparticle (LNP) conjugates have been developed to ensure higher efficacy in delivery and internalization of drugs; AAV gene therapy is used to deliver a copy of therapeutic genetic element to affected cells to rectify a defective gene, using the cell’s own transcriptional and translational machinery

modified. Phosphorothioate linkages within the ASO backbone have been incorporated to increase nuclease resistance, serum protein binding affinity and decrease hydrophilicity thus increasing the half-life of ASOs in body fluids. Several sugar modifications in ASOs have also been introduced. The presence of electronwithdrawing group at 2′-position of the ribose sugar in RNA results into a C3′-endo sugar puckering which is favorable for duplex formation. Thus, an RNA/RNA duplex is more stable than the corresponding DNA/DNA duplex. Thus, modifications in the sugar residue of ASOs have been introduced to get an RNA-like conformation. One of the most applied modifications in this platform is 2′-O-methyl (2′-O-Me), which is a naturally occurring modification (Fig. 4.2). Another important 2′-O-modification of ribose is 2′-O-methoxyethyl (2′-O-MOE), which increases binding affinity to RNA and resistance toward nuclease in vivo.

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Fig. 4.2 Chemical structure of modified and functionalized ASOs; To improve nuclease resistance and efficacy, the nucleobases, the ribose sugar moiety, the phosphodiester linkage of ASOs has been chemically modified. Some important modifications are 2′-O-Me, 2′-O-MOE, 2′-F-RNA, LNA, TNA, PNA, FANA, CENA, etc.

2′-O-aminopropyl (2′-O-AP) and 2′-fluoro modifications are also used resulting in improved RNA target binding affinity and apart from 2′-fluoro, increased nuclease resistance, except 2′-fluoro (Fig. 4.2). Nucleobase modifications are less common, but they can also be incorporated into ASO. Out of several applied modifications, only the replacement of cytosine with 5-methylcytosine has proved advantageous as

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Fig. 4.3 Mechanisms of silencing native ss-RNA; specifically designed gapmer, siRNA, artificial miRNA, and non-catalytic antisense methods are used to silence ss-RNA through RNA cleavage, splicing, decapping and deadenylation and block of translation, respectively

5-methylcytosine replacement decreases ASO immunostimulatory effects without compromising Watson–Crick complementarity. Sugar modifications are also applied to develop gapmer ASOs, where sequence with modifies sugar moiety is combined with an internal unmodified sequence (Evers et al. 2015) (Fig. 4.3). Another important modification platform in ASO chemistry is the development of locked nucleic acid molecules (LNAs), where the 2′ hydroxyl group of sugar molecule is attached with the 4′ carbon atom and tricycle-DNA oligonucleotides (Geny et al. 2016). Recently, another new class of backbone and sugar modifications adduct, called peptide nucleic acids (PNAs), has been developed, where sugarphosphate backbone is fully substituted by polyamide linkages (Wancewicz et al. 2010). PNAs also confirm resistance to degradation without compromising basepairing capabilities and binding affinity. tricyclo-DNA (tcDNA) and cyclohexenyl nucleic acids (CeNA) also showed wonderful biophysical features thus proving their potential to become new therapeutic candidate (Fig. 4.2). The choice of modified ASOs depends on the mode of action and the specific ASO chemistry selected for therapeutic target. Clinically approved ASOs mainly function through these two mechanisms: (1) RNAse H-triggered degradation of target RNA and (2) non-degradative steric hindrance of target RNA (Evers et al. 2015). ASOs with unmodified sugars can also activate RNAse H mediated degradation of target mRNAs efficiently thus resulting in gene silencing where RNAse H

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specifically cleaves the RNA moiety when binding with ASOs in mRNA-DNA heteroduplex. After that, ssDNA ASOs are released from the duplex and trigger degradation of additional mRNA molecules for a more efficient silencing (Fig. 4.3). This ASO chemistry has been optimized and thus widely used these days. This class of ASOs is currently the most widely used for therapeutic applications. MOE ASO gapmers, designed to target mutant SOD1, is the first in class example of ASOs approved for human clinical trial for the treatment of ALS (Ha and Kim 2014). Nusinersen, an antisense oligonucleotide (ASOs) already approved by the FDA to treat the patient of spinal muscular atrophy.

4.2.1.1 Ligand-Modified Small Interfering RNA Conjugates Small RNA molecules like siRNA (silencing RNA) and miRNA (microRNA) are involved in gene silencing pathway, known as RNAi pathway, thus regulating gene expression (Bobbin and Rossi 2016). siRNAs are produced from Dicer-mediated processing of dsRNAs, while miRNAs are produced from long primary transcripts (pri-miRNAs), through the classical Drosha/Dicer pathway or the splicing mediated mirtron pathway (Ha and Kim 2014). siRNA and miRNAs are loaded onto RNA-instructed silencing complexes (RISCs) mediating gene silencing through target-RNA cleavage by the help of Argonaute 2 (Fig. 4.5). This complex also recruits several epigenetic modifiers to change the epigenetic landscape which favors the silencing of a gene and the transcription machinery. miRNA and siRNA mediated gene silencing strategies have been extensively explored in therapeutic applications (Bobbin and Rossi 2016). Out of them, small hairpin RNA (shRNA) precursors based on the skeleton of endogenous pri-miRNAs found to be the best. Several chemical modifications have been performed to increase their stability. On the other hand, lentiviral and AAV vectors are used to deliver miRNAs and shRNAs into brain, making them a safer, non-immunogenic route of administration (Keiser et al. 2016). GalNAc-siRNA conjugates platform is an efficient way to increase the cellular uptake and target organ accumulation siRNA (Fig. 4.4). Many GalNAc–siRNA conjugates are currently under clinical trial to silence diseasecausing genes in hepatocytes and cardiometabolic disorders. Just like ASO therapeutics, backbone of siRNA molecule has also been modified chemically. Important modifications like 2′-OMe, 2′-fluoro and phosphorothioate linkages have been used in siRNAs to improve stability, binding affinity with target mRNA and decrease immunostimulation. Selective designing of the siRNA molecule is another important criterion to decrease off-target effects. To increase the RNAi efficiency and curtail down the off-target effects, glycol nucleic acid and altriol nucleic acid residues can also be incorporated to develop better RNAi conjugates. 4.2.1.2 Anti-miRNA Oligonucleotides (antagoMIR) Recent research showed that altered miRNA expression and function is one of the main reasons behind the manifestation of several diseases like cancer, metabolic disorder, neurodegeneration, and autoimmune diseases, thus making them a reliable therapeutic target. A single miRNA-based drug can alter the expression of several genes in that pathway at a time. Many strategies have been developed in the past

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Fig. 4.4 Schematic representation of a GalNAc–siRNA conjugate molecule; a trivalent ligand with terminal GalNAc moieties is covalently attached to siRNA at the 3′-end of the sense strand. GalNAc–siRNA conjugates platform is an efficient way to increase the cellular uptake and target organ accumulation siRNA

years to inhibit such disease-causing miRNAs in vivo. Downregulation of miRNA expression and function can be achieved by the development of anti-miRNA ASOs, small-molecule inhibitors of miRNAs and through expression of miRNA sponges. lncRNAs and pseudogenes can act as natural miRNA sponges (Cesana et al. 2011). MicroRNA (miRNA) sponges are RNA transcripts containing multiple high-affinity binding sites that associate with specific miRNAs and prevent them from interacting with their target mRNAs (Fig. 4.5). These miRNA sponges can reduce the overall concentration of active miRNAs. At present, the clinical application of miRNA sponges is limited. Another unique group of nuclease-resistant lncRNA is circular RNA (circRNA), which are mainly produced by covalent interaction between 5′ and 3′ termini, giving it a circular appearance (Memczak et al. 2013) (Fig. 4.5). circRNAs compete with protein-coding mRNAs thus regulating the expression. Small molecules designed to inhibit the expression of specific miRNA are also screened from libraries (Fig. 4.6). CiRS-7 is found to absorb miR-7, thus rescuing the target mRNA of a-synuclein (associated with Parkinson’s disease) from inhibition. But due to poor selectivity and high IC50, the therapeutic application of these molecules is limited. So, the success of this miRNA mediated therapy is coming from ASO platform. Single-stranded ASOs are being developed which can bind to the target miRNA with higher affinity and specificity, thus inhibiting their activity. These anti-miRNA oligonucleotides are called as antagoMIR or blockmir. Like ASOs, antagoMIR can also modulate RNA and protein expression by inhibiting endogenous miRNA. In the last decade, several miRNA-specific antagoMIRs have been designed and few of them are already in the phase I/II of clinical trials, for the treatment of cardiovascular diseases, metabolic disorders, liver cancer, etc (Li and Rana 2014). AntagoMIRs are also being used in mouse models for neurodegenerative disorders like AD, HD, PD, and ALS (Fig. 4.7).

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Fig. 4.5 Schematic representation of miRNA processing pathway; Most of the miRNAs are synthesized in the Dicer-dependent pathway. After transcription, the pri-miRNA transcripts are cleaved by Drosha in the nucleus, resulting into 60–70 nt long pre-miRNA. Then the pre-miRNA molecules are exported to cytoplasm by Exportin 5. After that, the hairpin region is sliced by Dicer to make ~22 nt miRNA duplex. After that, this miRNA duplex is separated into a guide and passenger strand. This guide strand is loaded with AGO protein to form RISC and targeted to specific mRNA. Artificial miRNAs (left panel) can also be designed by the replacement of mature miRNA sequence of a natural pri-miRNA for a complementary sequence of target mRNA. Then this sequence is being cloned in an expression vector and transfected in cell

4.2.1.3 Lipid Nanoparticles Due to the selective permeable nature of biological membrane, it restricts the entry of several charged and large molecules like nucleic acids. Lipid-based molecules like liposome are developed to overcome this. With the help of such lipid polymorphic phase, charged molecules like nucleic acids can easily pass through the cell membrane. Felgner et al. contributed heavily to the development of lipid-mediated delivery systems. Several lipid nanoparticle (LNP) conjugates have been developed to ensure higher efficacy in delivery and internalization of drugs (Samaridou et al. 2020). LNPs protect nucleic acid from serum nuclease, chemical, enzymatic degradation and immune cells thus increasing the half-life of the drug without triggering any immune stimulation. LNPs are composed of four components: phospholipids,

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Fig. 4.6 Roles of different RNA molecules used in the therapy of neurodegenerative disorders; messenger RNA (mRNA), microRNA (miRNA), exogenous or endogenous small interfering RNA (siRNA), circular RNA (circRNA), piwi-associated RNA (piRNA), and long ncRNA (lnc RNA)

ionizable cationic lipids, cholesterol, and polyethylene glycol (PEG)-lipids (Eygeris et al. 2022). The clinical application of LNP-based RNA therapeutics is mainly based on the development of ionizable cationic lipids, which can ensure more than 85% siRNA encapsulation, timely endosomal escape and maintain surface charge of LNP at physiological pH. Diffusible PEG-lipid design is also another crucial factor for clinical translation. Size of ionizable cationic lipids and PEG-lipid designing altogether dictate the clinical success of LNPs. LNPs are successfully used for gene silencing in hepatocyte cells through entrapping siRNA at a ratio of 0.095 (siRNA/ lipid). Lipid head groups are also modified by incorporating ionizable tertiary amine moieties, thus ensuring a net positive charge at acidic pH, decreasing cytotoxicity and immune stimulation. To avoid aggregation during LNP formation and to increase transfection efficiency and accumulation, diffusible PEG-lipids containing C14 alkyl chains were developed. Ethanol mixing technique is also used to mix

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Fig. 4.7 Current landscape of nucleic acid therapeutics against neurodegenerative diseases; AntimiRNA anti-microRNA, miRNA mimic microRNA mimic, RNase H: ribonuclease H, AO antisense oligonucleotide, lncRNA long non-coding RNA and siRNA small interfering RNA

preformed LNP with nucleic acids. Rapid-mixing techniques produced LNP– siRNA systems with high accumulation efficiencies (>85%) and narrow size distributions are also achieved through this rapid-mixing technique (Eygeris et al. 2022) (Fig. 4.8). Due to the small size, LNPs can easily cross the blood–brain barrier (BBB), thus making them a suitable therapeutic tool by permitting drugs to reach the damaged sites of the CNS in patients affected by neurodegenerative disorders, such as Alzheimer’s and Parkinson’s diseases (Hou et al. 2021).

4.2.1.4 Adeno-associated Virus Vectors Gene therapy is another rising therapeutic platform which is used to deliver a copy of therapeutic genetic element to affected cells to rectify a defective gene, using the cell’s own transcriptional and translational machinery (Porada et al. 2013). Gene

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Fig. 4.8 Schematic representation of an LNP containing siRNA or mRNA and important lipid components; LNPs are composed of four components: phospholipids, ionizable cationic lipids, cholesterol, and polyethylene glycol (PEG)-lipids. Lipid nanoparticle (LNP) conjugates have been developed to ensure higher efficacy in delivery and internalization of drugs. Ethanol mixing technique is also used to mix preformed LNP with nucleic acids. Rapid-mixing techniques produced LNP–siRNA systems with high accumulation efficiencies (>85%) and narrow size distributions are also achieved through this rapid-mixing technique

therapy can be implemented through many ways like using gene-editing technology, by using siRNA or miRNA to silence the gene, by the introduction of a functional copy of the gene (Saraiva et al. 2016). Prolonged, sustainable therapeutic effects of gene therapy made them advantageous. Gene therapy can be implemented through either viral or non-viral vectors. Although non-viral vectors show low immunogenic risk, very high therapeutic doses are required for efficient results. Thus, most of the clinically approved gene therapies are mainly dependent on viral vectors like adenovirus, lentivirus, herpes simplex virus (HSV), polio, adeno-associated virus (AAV), etc (Lundstrom 2018). Wild-type AAV is a small, non-enveloped parvovirus (~25 nm), having a ~4.7-kb ssDNA genome. All viral coding genes are altered with therapeutic genetic elements, without changing the capsid (Fig. 4.9). This attempt minimizes the packing volume and immune stimulation. At present, AAV is the most preferred vector for in vivo gene delivery. But unwanted genomic integration of viral vectors in target cells raises the alarm toward the viability and fate of this platform. So further, these viral vectors are recombined with suitable genetic elements to decrease their risk, thus making them more clinically suitable and effective delivery platform. Due to its broad range, AAV-based vectors can be targeted to diversified cells and tissues for transduction in a tissue selective way. This selectivity also ensures high-affinity

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Fig. 4.9 Schematic representation of an AAV vector containing a 4.7-kb ssDNA with inverted terminal repeats (ITR); Wild-type AAV is a small, non-enveloped parvovirus (~25 nm), having a ~4.7-kb ssDNA genome. All viral coding genes are altered with therapeutic genetic elements, without changing the capsid

binding with cell-specific receptor, thus leading toward endocytosis mediated cellular uptake. Several strategies like chimeric capsid engineering, viral pseudotyping have been applied to upgrade the tissue specificity and transduction efficiency. In this therapy, transgenes are transported directly to the cells, in vivo or ex vivo though recombinant AAV vectors (Porada et al. 2013). In the last few decades, several drugs which are based on recombinant viral-vector mediated delivery have been approved by FDA and EMA for the clinical trials of amyotrophic lateral sclerosis (ALS), spinocerebellar ataxia type 3 (SCA), Parkinson’s disease, Alzheimer’s disease, spinal muscular atrophy, Pompe disease, Huntington’s disease, etc (Hudry and Vandenberghe 2019). Several clinical trials have already showed that AAV-mediated therapy in human CNS is safe but after the delivery they can induce some off-target effects thus triggering some immune response. We need to predict the possible off-target outcomes and evaluate early during preclinical studies through in-depth bioinformatics screening, animal models testing, and iPSC technology.

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Strategies Involved in the Treatment of Neurodegenerative Disorders

4.3.1

Alzheimer’s Disease

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Alzheimer’s disease (AD) is the most common, dominant, irreversibly progressing and aging-related neurodegenerative disease, which accounts for 70–80% of all types of dementia and affecting nearly 50 million people and by 2050, the number of patients is predicted to triple. AD starts within the temporal area of the brain in the hippocampus and progresses to the cerebral cortex and other brain areas. AD pathology is exclusively marked by the extracellular, insoluble deposition of amyloid-β (Aβ) peptides in the form of senile plaques and intraneuronal neurofibrillary tangles developed by the hyperphosphorylation of tau protein. These aggregations finally lead toward chronic neuronal loss, cognitive impairment, progressing memory loss, and learning capabilities (Scheltens et al. 2016; Breijyeh and Karaman 2020). Till date, there is no absolute cure for AD. But recent approval of new drugs like cholinesterase inhibitors and N-methyl-D-aspartate receptor (NMDA) antagonists can improve neurocognitive function and reduce symptoms temporarily but they are unable to stop the progression of the disease. Till date, the molecular aspects of AD pathology are not clearly understood. But research in the last few decades pointed out some important proteins, having important roles behind the foundation of the disease. The manifestation of the disease is majorly dependent on the processing of amyloid precursor protein (APP) which is mainly dependent on amyloid-β precursor protein cleaving enzyme 1 (BACE1), leading toward the formation of Aβ fragments. Aβ1-40 and Aβ1-42, the main Aβ species aggregate to form plaques. Aβ1-40 and Aβ1-42 are mainly produced by the aberrant splicing of amyloid precursor protein (APP) by the action of β-site APP cleaving enzyme 1 (BACE1) and γ-secretase. On the other side, Aβ1-42 levels are increased by the mutation in the APP and presenilin (PSEN1) genes (codes for the catalytic subunits of γ-secretase), leading toward early onset of familial AD. Aβ oligomers promote cell death through synaptic loss and neuronal dysfunction (Breijyeh and Karaman 2020). Another important cause of AD is the intraneuronal neurofibrillary Tau tangles. Tau is a microtubule-associated protein expressed predominantly in neuronal axons and involved in the microtubule assembly and stability. The activity of tau is dependent on phosphorylation. But hyperphosphorylation of tau decreases the microtubule binding ability, thus leading toward destabilization of microtubules, reduced vesicular trafficking, and finally loss of synapse (Breijyeh and Karaman 2020). Hyperphosphorylation of tau is detrimental and directly connected with the neurodegeneration and the loss of neurocognitive coordination, termed “tauopathies.” So many recent therapeutic molecules are showing effective results in animal models, but they are falling flat in clinical trials due to the complexity of this disease. Thus, nucleic acid-based strategies can be the lifesaver and an effective alternative for the treatment of AD as they can be designed specifically to target wide range of

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pathological features. Hyperactivity of BACE1 is directly related with the foundation and progression of the disease, leading toward aggregation of Ab and neurodegeneration. Thus, BACE1 deactivation is being considered as a reliable, potential therapeutic target to treat AD. Several nucleic acid-based strategies have been applied to deactivate BACE1. Yan et al. developed two ASOs that can target BACE1 and found reduced release of Aβ40 and Aβ42 by 50–80% (Wolfe 2014). Splice-modulating ASOs were also designed by Wolfe et al to target BACE1, as alternatively spliced transcript variants at exons 2 and 3 do not show any β-secretase activity (Wolfe 2014). Significant reduction of Aβ production in cells was achieved without altering total BACE1 mRNA. McSwiggen and colleagues patented 325 BACE targeting siRNAs which includes a variety of chemically modified siRNAs, i.e. 2′-deoxy, 2′-F and 2′-OMe pyrimidine and purine nucleotides, phosphorothioate internucleotide linkages, etc (Chakravarthy et al. 2017). Most of these siRNAs successfully reduced the BACE expression by 40–90% at 25 nM concentration. Kao et al. also designed specific siRNAs, reducing BACE1 mRNA by more than 90% and Aβ production by 41%. Modarresi et al. also injected LNA-modified antisense transcript siRNAs targeting BACE1 into the third ventricle of Tg-19959 mice, leading toward downregulation of BACE1 antisense transcripts, BACE1 protein expression, less Aβ production, and aggregation in the brain (Modarresi et al. 2011). BACE1-targeting siRNAs were also injected into hippocampus of APP transgenic mice by lentiviral vectors, thus decreasing the concentration of BACE1, amyloid production, and neurodegeneration (Singer et al. 2005). Exosomes loaded with BACE1-specific siRNAs were injected into mouse brain, resulting into a significant downregulation of BACE1 mRNA and protein up to 60% in wild-type mice (Alvarez-Erviti et al. 2011). BACE1 downregulation is also achieved by specifically designed synthetic miRNAs in AD mouse model through adeno-associated viral particles mediated delivery, leading toward reduced plaque formation and disease progression (Piedrahita et al. 2016). Several miRNAs are dysregulated in AD brains, proving their nexus with AD pathology and the progression of the disease (Di Meco and Praticò 2016). BACE1 mRNA is a direct target of miR-188-3p, which is downregulated in the brain of AD mice, thus making it a therapeutic regulator. BACE1 levels have been reduced efficiently by overexpressing miR-188-3p through intracranial viral delivery, leading toward the improvement of neurological coordination, cognitive function. Nawrot et al. developed RNA-cleaving hammerhead ribozymes, which can downregulate BACE1 mRNA expression by more than 90% in HEK293 and SH-SY5Y cell line and reduce Aβ40 and Aβ42 production by more than 80%. In recent years, ASOs-based strategies have also been employed to turn down the expression of several proteins which are directly involved in AD pathogenesis. ASOs have been specifically designed and targeted to APP, PSEN1, and acetylcholinesterase, reducing disease pathology in transgenic AD mouse models (Farr et al. 2014; Fiorini et al. 2013). Ionis Pharma has patented (US 2003/0232435 A1) 78 gapmer ASOs with 2′-MOE wings which can target many regions of APP mRNA and inhibit APP protein expression in the range of 39–82%. Banks et al. also developed a radioactively tagged phosphorothioate DNA ASO targeting the Aβ region of APP, which

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can enter the cerebrospinal fluid of mice by crossing the blood-brain barrier (BBB). Higher dose of this ASO was administered in SAMP8 mice through intracerebroventricular injection, resulting into better learning capacity and enhancement in memory restoration. Peripheral injection of APP ASO to SAMP8 mice also resulted in a 30% increase in APP levels without any change in soluble Aβ levels, leading toward improved memory. ASO-mediated APP knockdown in Tg2576 mouse brains also reduced cytokine expression and improved learning and memory. Grilli et al. designed two phosphorothioate ASOs to target PSEN1 in wild-type mice and found lower PSEN1 expression, leading toward reduced cell death and better neuroprotection. Fiorini et al. also injected PSEN1 targeting ASOs to aged SAMP8 mice and found significant reduction in brain oxidative stress biomarkers (Fiorini et al. 2013). DeVos et al. screened 80 ASOs for tau knockdown. Out of them, three ASOs reduced tau mRNA levels by more than 75%. The best among them reduced brain tau mRNA and protein significantly in a dose-dependent manner while injected in mice (DeVos et al. 2013). Small-molecule (mitoxantrone) conjugated bipartite ASO and PNA-modified conjugated bipartite ASO have also been developed to target tau splicing (Devine et al. 2011). ASOs targeted against human acetylcholinesterase (AChE) mRNA also reduced AChE activity in an AD mouse model after 8 h with a lasting effect up to 42 h, accompanied by increased memory retention and improved behavior. miR-34c is upregulated in the hippocampus of AD patients and mouse AD models. Zovolis et al. designed specific miR-34c targeting antagoMIRs which rescued learning in mouse models. Lee et al. injected an miR-206 targeting antagoMIR, AM-206 into the third ventricle of Tg2576 mice, leading toward increased brain levels of brain-derived neurotropic factor, enhanced synaptic density in hippocampus, neurogenesis, synaptic plasticity, and memory. Several other antagoMIR are at present under preclinical trials for the treatment of AD. Nucleic acid aptamers have also been designed to inhibit Aβ fibrillation, BACE1 localization, and tau oligomerization to slow down the progress of AD. But still, detailed investigations are needed in future to establish aptamers as one of the clinically efficient therapeutic tools.

4.3.2

Parkinson’s Disease

Parkinson’s disease (PD) is an age-related, long-term, neurodegenerative brain disorder that triggers uncontrollable movements like shaking, limb rigidity, slowness in movements, mental health, thinking ability, meaning it causes parts of your brain to deteriorate. PD mainly affects predominately dopaminergic motor neurons in substantia nigra of our brain. Research shows that a-synuclein expression is directly related with the onset, progression, and risk of the disease. a-synuclein is overexpressed in this condition, triggering multiple phenomena to alter the homeostasis of the neuron. In rare cases, a-synuclein is found in aggregated states, which is a principal component of Lewy bodies. Enhanced level of wild-type a-synuclein, triggered by gene duplication and triplication event, can lead toward the manifestation of PD pathology (Devine et al. 2011). Recent research shows prion-like

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mechanism of a-synuclein. Thus, several approaches are being taken in the past few decades to inhibit the expression and the function of a-synuclein. ASO-based therapeutics has been developed to target a-synuclein mRNA. A 2-MOE ASO was designed to target LRRK2 mRNA for RNAse H-mediated degradation to improve pathologic α-synuclein inclusion-body formation in PD mouse model, thus reducing the levels of LRRK2 mRNA and protein with decreased aggregation of α-synuclein in the substantia nigra of treated mice. Phase 1 clinical trial (NCT03976349) of 2-MOE ASO has already started in PD patients. ASOs have been designed to target alternative splicing mechanism to change the ratio between the full-length α-synuclein transcripts and spliced isoforms, leading toward the development of a new PD therapeutics (Nakamori et al. 2019). Recently, another exciting one-step strategy has been designed to convert astrocytes to functional neurons in situ by RNase H-inducing ASOs. Fu’s group developed another phosphorothioate backbone-based ASO to knock down polypyrimidine tract-binding protein 1, thus converting primary astrocytes to dopaminergic neurons in the mouse brain, replenishing lost dopaminergic neurons and restoring striatal dopamine in a PD mouse model. Accumulation of α-synuclein protein and SCNA overexpression triggers the pathology of PD. So, siRNA mediated reduction of SNCA mRNA and protein levels has been considered as one of the reliable therapeutic strategies (Li et al. 2020). To silence SNCA expression, siRNA-protamine complex containing anionic liposomes ornamented with rabies virus glycoprotein (RVG) was injected in primary neuronal cells, leading toward significant reduction of α-synuclein protein in the hippocampal and primary cortical neurons of mouse. siRNA fused with RGV-exosomes has been administered into the brain of normal mice, leading toward the downregulation of a-synuclein mRNA and protein up to 50% in cortex, substantia nigra, and striatum. In transgenic mice with human phospho-mimic S129D a-synuclein expression has also been targeted with RSV-exosome-siRNAs, thus reducing the expression and aggregation of a-synuclein in the dopaminergic neurons of midbrain, leading toward delayed progression of PD in those mice. AAV-mediated delivery platform has also been used to downregulate the expression of a-synuclein. Recombinant AAV platform, which is used to deliver a-synuclein targeting shRNA in the substantia nigra of normal mice, resulting into 35% downfall of mRNA and protein levels of a-synuclein, without causing any further neuronal degeneration (Zharikov et al. 2015). AAV-mediated shRNA has also been proved to be a protective shield against dopaminergic neurons in the substantia nigra and in the striatum, when rats are exposed to PD like mimicking situations. shRNA mediated knockdown of α-synuclein in PD rat model was found to be neuroprotective by decreasing the degeneration of dopaminergic neurons (Li et al. 2020). Replacement of dopaminergic neuron loss has been achieved by converting astrocytes to dopaminergic neurons in a PD mouse model through the knockdown of polypyrimidine tract-binding protein 1. More preclinical investigations will be needed to make this approach more reliable and effective for PD. Nanoparticles like N-isopropylacrylamide combined with acrylic acid has also been used for efficient targeting and delivery of shRNA to the brain, by crossing blood–brain barrier. In addition, Nurr1 is another important protein that plays critical role behind the

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protection of dopaminergic neurons from neurotoxins and neuroinflammation (Dong et al. 2016). But in-depth research is needed to develop Nurr-1-based therapeutics for PD in future. Selective DNA aptamers have also been designed against α-synuclein oligomers, rather than monomers or fibrils, thus reducing the level of α-synuclein oligomerization and improving dopaminergic neuron loss. Administration of peptide-conjugated aptamers in SK-N-SH cells and primary neurons was found to be very efficient by inhibiting α-synuclein aggregation, promoting α-synuclein clearance, and providing protection against α-synuclein-triggered mitochondrial dysfunction (Zheng et al. 2018).

4.3.3

Huntington’s Disease

Huntington disease (HD) is a rare, fatal, inherited, autosomal dominant neurodegenerative disease that damages motor neurons with wide-ranging impact like affecting normal movement, mood, emotional problems, thinking, and neurocognitive ability. The mean age of the onset of HD is 35–44 years, and the median survival duration is 15–18 years after the onset. HD is mainly caused by the expansion of 36 or more CAG trinucleotide repeat in the first exon of the gene that codes for huntingtin (HTT) protein. Due to this expansion, the translated huntingtin (HTT) protein carries an elongated stretch of polyglutamines (polyQ) at the N-terminal, making it a mutated protein. This mutant protein is toxic which causes neuro-dysfunction and neurodegeneration, leading toward neurocognitive and psychiatric disturbances (Andrew et al. 1993). Still there is no permanent cure of this disease, but some recent therapeutic strategies can help to dilute some of the symptoms. Many therapeutic blueprints are currently under clinical investigation. But all these approaches raised the alarm on the possible off-target effects due to loss of normal HTT functions. Constitutive loss of wild-type allele is found to be lethal in mouse embryo, leading toward HD like phenotypes (Dragatsis et al. 2000). Recent reports showed that removal of HTT is non-deleterious in adult neurons. Being neuroprotective, wild-type HTT plays some pivotal role in different pathways (Saudou and Humbert 2016). Thus, several novel strategies have been taken to silence mutant HTT allele through nucleic acid-based technologies (Keiser et al. 2016; Beaudet and Meng 2016). Platforms like ASOs, RNAi silencing are leading from the front to tackle this situation, turning out to be promising by improving HD neuropathology. For the first time, knockdown of mutated HTT transcripts has been achieved through siRNA and shRNA mediated silencing in transgenic HD mouse models. Intra-striatal injections of cholesterol-conjugated siRNAs silenced mutant HTT transcripts effectively up to 70%, leading toward nearly 65% reduction in HD protein levels, thus decreasing the number and size of intranuclear inclusions and showing improved motor functions in mice. Specific siRNA molecules were designed to target HTT mRNA and reduction in the mRNA and protein level of HTT was achieved in HD models during preclinical trials. Effective silencing of mutant HTT was accomplished by the selective designing of siRNA and artificial miRNA, targeted to allele-specific polymorphisms of HTT gene in both patient-derived sample and mouse models

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(Monteys et al. 2015). AAV-mediated delivery of shRNA targeted against human mutant HTT, leading toward decreased mutant HTT mRNA and protein levels by 51–78% and 28–50%, respectively, decreased number of nuclear inclusions and improved behavior in HD mice. Significant reduction of lesion size and partial clearance of HTT inclusions were also recorded by expressing shRNA after the appearance of HD pathology. Inhibition of HTT protein expression has been achieved through specific designing of single-stranded and double-stranded ASOs and targeting them to patientsderived cells lines. HTT-ASOs has also been administered transiently into the cerebrospinal fluid of HD mouse models, causing suppression of HTR mRNA and proteins, resulting into decreased accumulation of mutant HTT protein and delayed progression of HD pathology. Similar consequences have also been seen in the case of nonhuman primates (Kordasiewicz et al. 2012). Selective downregulation of HTT was also achieved by designing allele-specific single-nucleotide polymorphic ASOs (Southwell et al. 2014; Kay et al. 2015). AAV-based platform was also used to inject anti-HTT shRNA into the striatum of HD transgenic mice, leading toward noteworthy downfall in the level of mutant HTT mRNA up to 55%, along with reduction of HTT pathology and improvement in the motor neuron mediated coordination. Peptide nucleic acid (PNA) peptide conjugates and locked nucleic acid (LNA) oligomers were also found to be effective to inhibit allele selective mutant HTT expression in HD human cell lines. CAG repeats targeting PNAs and LNAs are more effective than the inhibition by a deletion polymorphism targeting siRNA. Better efficacy of these siRNA mediated strategies has been achieved during preclinical studies by using adeno-associated vectors (AAV), lipid nanoparticles, and cholesterol-conjugated molecules (Keiser et al. 2016). For effective silencing of HTT in the mice brain, one single intravenous injection carrying modified AAV that encodes an artificial miRNA was found to be adequate (Choudhury et al. 2016). Right now, several new AAV-based platforms are under trails, which can lead toward the formulation of next generation silencing therapy against diseases like HD. According to a report, silencing of nonallele-specific HTT by ASOs has already got into Phase 1/2 clinical trial. Several other approaches are also being developed to quantify the concentration of mutant huntingtin protein in the cerebrospinal fluid (Southwell et al. 2014; Rodrigues and Wild 2020). The pharmaceutical company, uniQure, already started a phase 1/2 trial in HD patients to evaluate the safety and efficiency of a one-time therapy, AMT-130. AMT-130 consists of an AAV5 vector with an artificial microRNA, designed to silence both mutant and wild type HTT genes. (ClinicalTrials.gov: NCT04120493) (Rodrigues and Wild 2020). This clinical trial is the very first attempt using AAV-mediated gene therapy for the treatment of HD. Recent research reported the neuroprotective nature of brain-derived neurotrophic factor (BDNF), making it another therapeutic target for HD. The progression of HD and the severity of motor and cognitive functions in HD mouse models are regulated by BDNF. Mutant HTT protein binds to the BDNF promoter and reduces the transcriptional activity in the cerebral cortex. BDNF has been found in lower level in the cerebral cortex and striatum of HD patients and in mouse models. AAV vector

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mediated delivery of BDNF provided neuroprotection to striatal neurons in rat model and improved motor dysfunction (Zuccato et al. 2011). Several strategies were also taken to restoring the transcriptional balance and cell metabolism in HD. Huntingtin protein interacts with several transcription factors like TATA-binding protein (TBP), Sp1, p53, and cAMP-response element binding (CREB) binding protein. Thus, mutant huntingtin protein can dysregulate the transcriptional output. Repressor element 1 silencing transcription factor (REST) is a general repressor of neuronal gene expression and is dysregulated in HD. Impairment of REST regulated gene expression is one of the preliminary events behind HD pathogenesis. This made REST another new therapeutic target. Double-stranded oligonucleotide decoys have been designed to target DNA binding region of REST, thus overriding the transcriptional activity, epigenetic repression mediated by REST and rescuing its target mRNA and protein levels in HD cell model (Li and Li 2004).

4.3.4

Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is an autosomal recessive, inherited, childhoodonset, motor neuron neurodegenerative disorder characterized by the loss of motor neuron, muscle weakness, limited mobility, breathing problems, and atrophy. SMA induced mortality is very high among infants, with a probability of 1 in 6000 live births all around the globe. SMA is mainly caused by the loss of function mutation or deletion in the survival of motor neuron 1 (SMN1) gene, resulting into the reduced level of survival motor neuron (SMN) protein and motor neuron degeneration. SMN protein is a very important protein of spliceosome subunit biogenesis, which is associated with the disease pathology of SMA. SMA severity can be modified in humans due to the presence of a duplicate gene SMN2, which is similar to SMN1 (Shababi et al. 2014). At present, SMA therapeutics is the only clinically efficient splicing correction therapy in humans. Transcription and translation of normal SMN protein can be restored by tuning the mRNA splicing of SMN2 gene by using ASOs. In-depth basic and translational research, associated with clinical studies paved the way to get the approval of SMA therapies from FDA (Food and Drug Administration, USA) and EMA (European Medicines Agency, EU). All the approved SMA therapies were developed on the same strategy so that they can increase SMN protein expression. Till date, two nucleic acid-based therapies have been approved, i.e., ASO-based modulation of SMN2 spicing (Nusinersen/Spinraza) and AAV9 mediated gene therapy to introduce an intact additional SMN1 cDNA copy (onasemnogene abeparvovec/Zolgensma). After getting satisfactory results from children with SMA, Spinraza (Nusinersen) got the final approval from FDA in December 2016, and from EMA in June 2017, becoming the very first genetic treatment for SMA (Table 4.1). Nusinersen is an 18-mer ASO, chemically modified by 2′-O-2-methoxyethyl phosphorothioate to get protection from nuclease degradation and is designed to target SMN2 gene by inhibiting the binding of hnRNP A1 to the intronic splicing silencer N1 element of intron 7 of SMN2 gene, thus disrupting a splice inhibitor site and promoting the

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inclusion of exon 7 in the SMN2 pre-mRNA. This ASO increases SMN protein levels by modulating the splicing of SMN2 pre-mRNA. In successive phase 3 clinical trials, infants with later-onset SMA were injected with intrathecal administration of nusinersen, so that it can be taken up by motoneurons from the cerebrospinal fluid (CSF). Improved motor function and reduced risk of death were reported in nusinersen-treated infants (Duong et al. 2021). SMN1 gene therapy is another way to increase the levels of SMN protein in motor neurons and in other cell types. SMA is an autosomal recessive disorder, caused by the loss of function of the SMN gene, thus making it a suitable target for gene therapy mediated by AAV vectors. A self-complementary, non-replicating AAV9 vector is loaded with the copies of SMN1 cDNA and delivered systematically to the CNS and spinal motor neurons to restore the SMN protein levels. Preclinical studies performed in a mouse model of SMA confirmed the upregulated expression of SMN protein, through AAV9 mediated gene transfer in peripheral tissue and motor neurons. Significant enhancement of life span up to 250 days has been reported in the SMA mouse model, which were treated with scAAV9-mediated SMN1 gene therapy. This AAV vector mediated gene delivery system for SMN1 is termed onasemnogene abeparvovec, commonly known as AVXS-101 and marketed under the brand name Zolgensma, developed by the Swiss drugmaker Novartis, becoming the first gene therapy to be approved by the FDA on May 24, 2019, and later by the EMA for the treatment of pediatric SMA patients up to 2 years of age. A single dose of 1.1 × 1014 vector genomes (vg) per kilogram (kg) body weight was recommended by FDA and was found to be enough to induce SMN protein expression throughout the CNS and peripheral organs. The dose was found to be well tolerated in SMA type 1 or 2 patients and pre-symptomatic SMA infants, making it a successful therapy for the treatment of SMA. A single intravenous injection of onasemnogene abeparvovec during phase 1 clinical trial resulted in expansion of life span in patient with infantile-onset SMA. Efficacy and safety of onasemnogene abeparvovec has also been evaluated in two successive phase 3 clinical trials (Table 4.1) (Mendell et al. 2021). The treatment is going to cost $ 2.125 million thus making it the costliest drug ever to be marketed (Mahajan 2019).

4.3.5

Frontotemporal Dementia (FTD)

Frontotemporal dementia (FTD) is a common term used for several brain disorders that primarily affect the frontal and temporal lobes of the brain, causing the lobes to shrink and affective movement, behavior, and personality. Several hexanucleotide (GGGGCC) repeat expansions were found in the first intron of C9orf72 gene, which identified as one of the main reasons behind FTD and ALS. Recent research shows that neurodegeneration in FTD mainly arises due to the loss of function of C9orf72 gene or RNA toxicity. RNA foci are also found in FTD/ALS brain. ASO-mediated therapy has already been reported by several groups to inhibit C9orf72 expression, the development of RNA foci (Donnelly et al. 2013; Sareen et al. 2013). Specific ASOs have been developed and targeted to decrease the toxic repeat expansion in

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patients-derived iPS cells, iPS-differentiated to motor neurons and patients’ fibroblasts. ASOs were specifically designed to target the transcript having hexanucleotide repeats. Downregulation of C9orf72 transcript formation and RNA foci suppression has been achieved in patient-derived cells through ASO-mediated therapy. Only antisense-specific ASOs have been selected as therapeutic tool, as sense-specific ASOs were unable to hybridize with the target. On the other hand, siRNA mediated therapy has also been designed to target C9orf72, but surprisingly there was no effect, making ASO-mediated platform more reliable and clinically advantageous. Effective downregulation of C9orf72 was observed when C9orf72specific ASOs were administered in mouse brain through intra-cerebroventricular stereotaxic injection (Lagier-Tourenne et al. 2013). Prolonged downregulation of C9orf72 mRNA (up to 40%) was confirmed by microarray analysis without any behavioral and neuropathological abnormality. Still c9orf72-mediated pathology and disease mechanism is not clearly understood. Research is going on to find out key regulators, involved behind the onset and the progression of the disease, which can lead toward the identification of new and clinically effective therapeutic target in future.

4.3.6

Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease (MND) or Lou Gehrig’s disease, is a fatal, progressive, neurodegenerative disorder triggered by the selective loss of motor neurons in the spinal cord and brain that control voluntary muscles. Early symptoms of ALS are dysregulated muscle function, severe muscle weakness, muscle twitching, and muscle stiffness. But, as ALS progresses, it eventually causes paralysis and early death mainly by respiratory failure. In almost 95% cases, no cause has been characterized yet for this disease, called as sporadic ALS. Rest of the 5% is genetically connected with the family tree. Both environmental and genetic factors are involved behind the onset and progression of the disease. Being rapid and progressive, ALS gets worse over time (Zarei et al. 2015). Most of the patients survive maximum up to 3–5 years after the diagnosis. Though there is no cure of this disease, several advanced therapeutic strategies have been taken to slow down the progression of ALS. In the recent years, superoxide dismutase 1 (SOD1) became one of the most reliable targets to treat ALS. SOD1 is a mutated protein found in the familial cases of ALS. In rat disease model expressing human mutated SOD1 (SODG93A), SOD1 specific ASOs were administered into the cerebral ventricles and proved to be successful in reaching spinal cord traveling through the cerebrospinal fluid. Delivery method for ASO has been optimized and the efficacy of the strategy has been evaluated in above model. SOD1-ASO treated mice showed significant downregulation in the level of SOD1 mRNA and protein across the spinal cord and brain, resulting into slower disease progression and higher survival rate. Later, SOD1-ASO got the approval of phase-3 clinical trial in the patients with different types of SOD1 mutation. Samples were taken from ALS patients and analyzed. Higher ASO level was recorded from the

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injection site as compared to another distant region. ASO peak concentration was observed after 12 h of infusion and gone down to the basal level at 24 h. A variation of 15% was observed in SOD1 levels from cerebrospinal fluid, forecasting the need of higher doses of drugs to achieve effective downregulation of SOD1 (Miller et al. 2020). siRNA-based antisense strategy has also proved its effectiveness against ALS in SODG93A animal models. siRNA molecules are mainly administered as shRNA with the help of viral vectors. Several siRNA silencing-based studies in ALS mouse models have already confirmed downregulation of SOD1 expression, delayed manifestation of the disease, and improvement in the motor functions (Wang et al. 2008). Based on recent findings, miR-155 became another therapeutic hotspot to treat ALS. miR-155 was found to be upregulated in SODG93A mice and in the spinal cord of ALS patients. ASO-based therapy has been devised to inhibit miR-155 (antagoMIR) and intra-ventricular administration in mice showed slow progression of the disease and extended survival (Koval et al. 2013). The therapeutic potential and reliability of this miR-155 targeted antagoMIR have also been evaluated through transcriptomics and proteomics studies in SODG93A transgenic mice (Butovsky et al. 2015). LNA-based preparation of anti-miR-155 was also injected in SOD1 mice through intra-ventricular route, leading toward delayed onset and progression of the disease (Butovsky et al. 2015). In the last few years, hexanucleotide (GGGGCC) repeat expansions found in the C9orf72 gene have emerged as a prime cause of ALS, frontotemporal dementia (FTD), and the amalgamation of ALS and FTD (ALS-FTD). These expansions are also found in few patients with sporadic ALS too. Though the exact mechanism of disease pathogenesis by this abnormal repeat expansion is not properly understood, the loss of normal C9orf72 function and gain of RNA toxicity have been identified as one of the main causes behind the pathogenesis. ASO-mediated therapy has already been targeted to neurons and fibroblast of C9orf72-related ALS patients and mouse model expressing the expanded C9orf72 gene. But even after the binding of ASOs with the C9ORF72 mRNA transcript or the repeat GGGGCC expansion, there was no reduction in RNA levels. However, RNA toxicity has been effectively reduced by ASOs, leading toward the protection against glutamate toxicity, which is another crucial factor behind the pathophysiology of ALS (Mathis and Le Masson 2018). To achieve better therapeutic efficacy, ASOs need to target both sense and antisense RNA foci, as both these strands of C9orf72 are transcribed in the fibroblasts from patients with the hexanucleotide repeat expansions. Finally, significant improvement of behavior in mice has been reported even 6 months after the single dose of ASOs, targeting repeat containing RNA. According to latest findings, C9orf72 mediated pathogenesis is driven by an anomalous splicing mechanism in tissues, leading toward another new therapeutic window to tackle the situation. Polyglutamine (polyQ) tract expansions and ATXN2 gene (codes for the Ataxin-2 protein) mutation are another important factor behind ALS progression (Elden et al. 2010). Ataxin-2, an RNA-binding protein plays several pivotal roles in the assembly of stress granule, RNA metabolism, concentrating aggregation prone proteins like TDP-43. The aggregation of pathological proteins in ALS is induced by TDP-43 (Prasad et al. 2019). Ubiquitinated cytoplasmic

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inclusions of TDP-43 became one of the important disease markers associated with 97% of ALS patients and 50% of FTD patients. Mutation in the TARDBP gene, that codes for TDP-43, is also found to be associated with some of the familial and sporadic cases of ALS and FTD. TDP-43 mediated toxicity is suppressed by the decrease in Ataxin-2. Thus, Ataxin-2 plays a very important role behind the onset of the disease. PolyQ expansions in Ataxin-2 are therefore considered as a measure of risk factor for ALS, thus placing them as another therapeutic target. ATXN2 targeted ASOs has been designed and administered to CNS of TARDBP transgenic mice, leading toward improved motor functions and better lifespan (Nizzardo et al. 2016; Amado and Davidson 2021).

4.4

Conclusion

A variety of potential genetic regulators in nexus with environmental factors and natural aging cumulatively contribute to the onset and the progression of these complex multifactorial neurodegenerative diseases. Unraveling the complex landscape of these disorders already provided few important therapeutic targets. Though current therapeutic candidates are showing effective results in cell lines and animal models, they are not being able to cross all the phases of human clinical trials. This disappointing result might have arisen due to targeting one cause at a given time. Being complex in nature, several targets relevant to these disorders converge with other neurodegenerative pathways. The primary trigger of neurodegeneration is different in all the diseases, but downstream pathways are shared in many cases, thus leading toward the reliance on one well-defined specific target for the treatment of genetically distinct monogenic diseases like ALS and HD. But for the treatment of complex neurodegenerative disorders like AD and PD, multiple targets should be aimed simultaneously to get better efficacy. Due to its ability to target multiple factors at a time, nucleic acid-based therapeutics became advantageous and reliable. Developing efficient therapeutic strategy is challenging because the disease pathology is regulated by so many diverse factors like genetics, environmental exposure, diet, and metabolism. All these existing treatments are trying to slow down the progression by treating the symptoms only, not going against the main underlying cause. Thus, there is an unmet demand of better nucleic acid-based therapeutics. In comparison with the conventional protein targeted small-molecule drugs, nucleic acid-based therapeutic agents like ASOs, siRNAs, miRNA, antagoMIRs, DNAzymes, and aptamers can selectively modulate the expression of important proteins by targeting their mRNAs (Fig. 4.7). Nucleic acid-based therapeutics hold a great promise for developing novel strategies to treat these complex neurodegenerative disorders as they can target a broad range of neuropathological features and it is easier to deliver nucleic acid-based drugs through the blood–brain barrier than conventional drugs. Present clinical status of several nucleic acid-based therapeutics against neurodegenerative disorders is shown below (Table 4.1). But still, we need to optimize the invasiveness of delivery methods and better administration protocol, to get long-term effect and benefit from these therapies. Few

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Fig. 4.10 Routes of administration for approved in vivo nucleic acid therapeutics; Intravitreal, intrathecal, and subcutaneous administration of ASOs can regulate pre-mRNA splicing and induce RNAse H1- or interfering RNA (RNAi)-dependent transcript degradation; intramuscular administration of LNP-conjugates can elicit potent immune responses; subretinal injection of AAV vectors is administered for the treatment of retinal disorders; AAV vectors-based drugs can also be administered intravenously

nucleic acid-based drugs have already got the approval from FDA, showing the potential of these drugs as next generation therapeutics to treat neurological diseases. The main hurdle remains in our inability to understand and discriminate between the primary and secondary disease mechanisms. Thus, the complex molecular mechanisms behind the onset and the progression of these diseases are not well understood till date. Integrated research can only fill the missing spot through the identification of new, clinically relevant targets. On the other hand, delivery platforms and administration processes also need to be improved for better efficacy (Fig. 4.10). Neuroinflammation is another significant component behind the

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neurodegeneration and disease pathology. Several nucleic acid-based strategies have also been taken to treat neuroimmune disorders. Currently nucleic acid-based drugs are also being used for neuro-oncology and trauma management, thus taking this area of research to a new dimension. Thus, the pace of nucleic acid-based therapeutics for the treatment of neurological disorders will surely accelerate in upcoming decades and will have a revolutionary impact on many diseases with no or limited treatment options.

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Human Diseases Induced by Oxidative Damage in DNA Suman Panda, Oishika Chatterjee, Gopeswar Mukherjee, and Subhrangsu Chatterjee

5.1

Introduction

We are continually exposed to many environmental carcinogens. Few carcinogens show specific toxicity via the development and enhancement of cellular levels of reactive oxygen species (ROS), such as superoxide anion and reactive nitrogen species (RNS). Reactive oxygen and nitrogen species (RONS) are produced by several endogenous and exogenous processes. On the other hand, there are several antioxidant defense processes to scavenge them. Due to the imbalance between RONS production and these antioxidant defenses, oxidative stress arises. Reactive oxygen species (ROS) are continuously produced during metabolic reactions in all the living cells. Out of them, singlet oxygen, superoxide anion, hydroxyl radical, and hydrogen peroxide are widely known ROS (Sharifi-Rad et al. 2020). Human signal transduction pathways are regulated by the pool of endogenous ROS. The lifespan of our cells depends on the balance between the generation and degradation of ROS. The antioxidant system of our body maintains the redox equilibrium thus removing excess free radicals. The antioxidant systems are composed of simple antioxidant molecules like vitamin C and E, which can scavenge free radicals and prevent biomolecules from damage. There are some enzymatic systems which also act as antioxidant systems which includes superoxide dismutase (SOD), catalase, glutathione peroxidase etc. Together these system works to maintain the concentration of ROS within a limit (Melis et al. 2013) (Fig. 5.1). Due to the exposure of different exogenous factors, ROS concentration increases inside cells, leading toward redox imbalance. ROS can induce extensive oxidative

S. Panda · O. Chatterjee · S. Chatterjee (✉) Department of Biophysics, Bose Institute, Kolkata, West Bengal, India G. Mukherjee Barasat Cancer Research and Welfare Centre, Kolkata, West Bengal, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 S. Chatterjee, S. Chattopadhyay (eds.), Nucleic Acid Biology and its Application in Human Diseases, https://doi.org/10.1007/978-981-19-8520-1_5

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Fig. 5.1 Activators and inhibitors of reactive oxygen species (ROS) production. Activators like chemotherapy, radiation, growth factors, cytokines, and hypoxia induce the formation of ROS. Antioxidant systems like SOD, catalase, glutathione, peroxidase, and thioredoxin reductase scavenge ROS to maintain the redox equilibrium in our cells

Fig. 5.2 Consequences of DNA damage induced by oxidative stress. Oxidative stress induces wide arrays of DNA damage, including double strand break (DSB), single strand break (SSB), oxidation of specific bases (8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) and 8-oxo-7,8dihydroguanosine (8-oxo-G)), and mismatched base. Abnormalities in DNA repair pathways lead toward genome instability, altered cellular equilibrium, or cell death

damage to all the biomolecules like nucleic acid, lipids, and other important cellular structures, leading toward necrosis, mitochondrial dysfunction, synaptic deficit, cognitive impairment, and metabolic disorders (Cadet and Davies 2017) (Fig. 5.2).

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If not repaired, ROS induced damage resulted in several diseases. Oxidative stress is directly involved in several age-related diseased conditions, i.e., cancer, neurodegenerative diseases, cardiovascular diseases, chronic obstructive pulmonary disease, and chronic kidney disease. Oxidative damage to nucleic acid can cause diverse mutations like deletion, addition, and base substitution. Many oxidized adducts of nucleic acids are generated due to this oxidative damage. Among all types of oxidative products, 8-Oxo-7,8- dihydro-2′-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxo-G) are widely studied predominant ROSinduced oxidative products. In this chapter, we discussed the nexus between oxidative nucleic acid damage and manifestation of various diseases. Finally, we also discussed the potential of oxidized biomarkers in disease diagnosis and the development of antioxidant therapy.

5.2

Types of Oxidative Damage

5.2.1

Formation of 8-Oxo-G

ROS can induce a wide variety of DNA damage in different DNA bases. But due to the low redox potential, guanine (G) is particularly susceptible to singlet oxygen, thus vulnerable to oxidation, leading toward the formation of different species of oxidized G products in DNA as well as in the RNA level. Among all the different oxidized DNA lesions generated by ROS, 7,8-dihydro-8-oxo-2′-deoxy-guanine or “8-oxo-G” is the most abundant and best studied lesions (Slupphaug 2003). 8-oxo-G is produced by the incorporation of an oxo group on the carbon 8 (C8) and a hydrogen atom to the nitrogen at position 7 (N7) of deoxy-guanine. Under physiological circumstances in normal tissues, 8-oxo-G lesions are found up to 103 lesions per cell, which can be increased in cancer cells (Suzuki and Kamiya 2017). For that reason, levels of 8-oxo-G are used as an important biomarker to measure the level of oxidative stress of individual cells or tissues. 8-oxo-G lesions are also marked as a risk assessment factor during the diagnosis of many diseases like cancer, neurodegenerative diseases, and cardiovascular diseases. When 8-oxo-G is present in DNA, all the cells face the main challenge during S-phase when they must replicate their genome with identical sequence and information. But 8-oxo-G does not act as a blocking lesion, like other DNA lesions, to stall the progression of the replication fork during replication. 8-oxo-G functionally mimics as a thymine (T) base in syn conformation leading toward a stable formation of a mutagenic A(anti):8-oxo-G (syn) mismatch pair instead of the non-mutagenic C(anti):8-oxo-G(anti) base pair (Fig. 5.3). As a result of this stable Hoogsteen base mispairing, replicative DNA polymerases incorporate the incorrect A opposite 8-oxo-G instead of the correct C frequently. As 8-oxo-G mimics a cognate base pair, A:8-oxo-G mismatch pair escapes the proofreading activity of polymerases. But the correct C:8-oxo-G base pair is recognized as a mismatch, thus leading toward lower efficiency of C incorporation opposite 8-oxo-G, resulting in C:G → A:T transversion mutation in

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Fig. 5.3 (a) Oxidation of guanine to 8-oxo-guanine leads to structural change of guanine. (b) 8-Oxo-guanine(syn) interaction with adenine(anti), anomalous guanine-adenine pairing, leading toward GC to AT transition mutation

the DNA of daughter cells if not corrected (Cadet and Davies 2017; Poetsch 2020) (Fig. 5.4).

5.2.2

Formation of 8-Nitro-G

8-nitroguanine (8-NO(2)-G) is another well-studied oxidative DNA adduct present either in DNA/RNA or in free form. The major sources of reactive nitrogen species (RNS) are peroxynitrite, the myeloperoxidase-H(2)O(2)-nitrite system, etc. (Hu et al. 2018; Ohshima et al. 2006) (Fig. 5.5). Increased formation of 8-NO (2)G has been reported in various diseased conditions like pancreatic cancer, Helicobacter pylori-induced gastritis, and RNA virus-induced pneumonia in mice (Hiraku 2010). Reactive nitrogen species has been found in inflammatory cells and tissues but not reported in normal cells. 8-NO (2)-G in DNA is a potent mutagen, resulting in G:C to T: A transversion during replication. While present in RNA, 8-NO (2)-G interferes with RNA transcription and modification. Mediated by various reductases, nitrated guanine nucleosides and nucleotide contribute to oxidative stress via production of superoxide. They also modulate various important enzymes like GTP-binding proteins and cGMP-dependent enzymes directly (Ohshima et al. 2006). Carcinogen which can induce pancreatic cancer has not been discovered to date. On the other hand, pancreatic cancer, especially hereditary

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Fig. 5.4 Mutations induced by the formation of 8-oxo-guanine. 8-oxo-G lesion can be formed in DNA by 2 different mechanisms. (a) Reactive oxygen species (ROS) can directly oxidize guanine in DNA; 8-oxo-G functionally mimics as a thymine (T) base in syn conformation leading toward a stable formation of a mutagenic A(anti):8-oxo-G(syn) mismatch pair instead of the non-mutagenic C(anti):8-oxo-G(anti) base pair. During replicative DNA polymerases incorporate the incorrect A opposite 8-oxo-G instead of the correct C frequently. If not repaired, a second round of replication will lead toward the formation of a A: T base pair. A:8-oxo-G mispairs can give rise to C: G → A:T transversion mutations. (b) ROS can also induce the oxidation of free dGTP in the nucleotide pool, thus forming 8-oxo-dGTP (o dGTP), which can be incorporated opposite A to form an A:8-oxo-G base pair

pancreatic cancer, has always been found in a nexus with acute and chronic pancreatitis. Pancreatitis is mainly inflammation of the pancreas. To eradicate harmful microbes and dead cells, pancreatic inflammation invites neutrophils and phagocytic macrophages and secretes nitric oxide (NO) and superoxide (O2˙) in a large amount. Due to the unstable nature of NO and O2˙, they react with each other to generate peroxynitrite (ONOO-) at the site of inflammation. ONOO- is not a potent free radical itself. ONOO- reacts with bicarbonate (HCO3-), secreted from pancreatic acinar cells in high concentration to generate nitrosoperoxycarbonate (ONOOCO2-), which is a major reactive nitrogen species. (RNS) in vivo that ionizes to form carbonate radical (CO3˙ -) and nitrite (NO2˙). Both the free radical CO3˙ - and NO2˙ are very detrimental to the structural integrity of DNA. As deoxyguanine (dG) has the least redox potential, it readily gets oxidized by CO3˙ - and NO2˙ (Ohshima et al. 2006).

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Fig. 5.5 Types of oxidized guanine species. 8-Hydroxyguanine, a ribose free base, is found both in DNA and in RNA, while 8-hydroxyguanosine lesions are mainly confined to RNA and 8-hydroxy2‘deoxyguanosine is confined to DNA. 8-Nitroguanine is another well-studied oxidative DNA adduct present either in DNA/RNA or in free form

5.3

Oxidative DNA Damage Repair Systems

5.3.1

Base Excision Repair (BER) Pathway

The frequency of oxidative DNA damage depends on the balance between the overall impact of the damage and different modes of DNA repair mechanisms, which can be regulated by different factors. As guanines are readily oxidized in DNA; thus, 8-oxo-G is estimated to occur on average about 100 to 500 times in DNA of each human cell per day. Oxidation of guanine also takes place in nucleotide pool as 8-oxo-dGTP and is then incorporated into DNA during replication. Microarrays and pull-down assays showed that 8-oxo-G is mainly found in gene deserts, adjacent to perinuclear regions of the heterochromatin. The perinuclear position of heterochromatin is heavily exposed to oxidative stress, thus leading toward increased

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oxidative DNA damage. But no sequence specificity has been found with the higher accumulation of 8-oxo-G heterochromatin (Maynard et al. 2008). Base excision repair (BER) pathway is the prime mechanism to repair 8-oxo-G adduct and other base modifications. 8-oxo-G is mainly excised by a special enzyme 8-oxo-guanine DNA glycosylase (OGG1) leaving an apurinic site (AP site). As the half-life of 8-oxo-G is only up to 11 min, so making the first step of repair is very efficient. Further apurinic sites are processed into single strand breaks via AP-endonuclease 1 (APE1) through backbone incision (Lee and Kang 2019). In case of long patch base excision repair, the damaged base and some additional nucleotides are replaced by the synergistic activity of eukaryotic polymerase delta (Poldδ) and epsilon (Polε) with the help of proliferating cell nuclear antigen (PCNA). Flap-endonuclease 1 (FEN1) removes the old strand and then ligase I (LigI) ligates the backbone together. On the other hand, in short patch base excision repair single base is replaced by polymerase beta (Polβ), and then ligated by ligase III (LigIII). X-ray repair cross-complementing protein 1 (XRCC1) also aids in this process as a scaffold for additional factors (Van Houten et al. 2018) (Fig. 5.6). However, there is a very little amount of information about this mechanism as the pathways are not well understood. With the help of different glycosylases, a wide variety of base modifications are repaired through BER leaving AP (apurinic/ apyrimidinic) sites as repair intermediates. Spontaneous de-pyrimidination and de-purination can produce AP sites, which is found about 2000–10,000 times in the genome per day. Under physiological conditions, a fraction of AP sites are derived from oxidative DNA damage and further processed by APE1. Few thousand 8-oxo-G sites and ~15,000 to ~30,000 AP sites per cell are found because of the balance between the rate of the formation of new DNA lesions and the efficiency of DNA repair enzymes. AP sites are mainly found in repeats and retrotransposons which can be activated during DNA damage by ionizing radiation (Lee and Kang 2019). Due to scientific limitations, these numbers may not be accurate, but it clearly signifies the importance of this mechanism and the downstream effects. Oxidative DNA damage is formed in a tissue-selective manner most of the time as our body tissues are exposed to different types of oxidative DNA damage because of their location in our body and enzymatic and metabolic activity. For this reason, all the tissues show different ways to deal with oxidative stress through specific DNA repair pathway, selective enzymes, and other protective mechanism. For this reason, oxidative DNA damage is highly variable between different cells and tissue types. The magnitude of this variability and the regulatory mechanisms in nexus are still not properly understood. Most of the DNA oxidative damage is repaired by base excision repair (BER) pathway, but few lesions escaped from the proofreading activity of repair systems, leading toward single-stranded (SSB) or double-stranded breaks (DSB). There are specialized mechanisms to repair these SSBs and DSBs. But in the case of complete absence of DSB repair systems or with low fidelity output, this damage ultimately leads toward genomic instability, unwanted chromosomal fusion, and fragile telomere. If unrepaired, then in BRCA2-dependent homologous recombination, these

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Fig. 5.6 Base excision repair (BER) of 8-oxo-G. Oxidative DNA damage can be repaired by base excision repair (BER) through several steps. A reactive apurinic site (AP site) is formed after the removal of the oxidized base by the action of OGG1. Then, single strand break is developed through incision by APE1. Finally, the damaged site is then repaired through either short or long patch BER pathway by the action of polymerases and ligated by ligases

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lesions induce severe genomic instability in the pancreas and breast and then induce tumorigenesis. Recent research has shown that excessive formation of reactive nitrogen species (RNS) in the pancreas induces hereditary pancreatic cancer in the absence of BRCA2-mediated repair. Suppression of RNS by antioxidant reduces DNA lesions and delays the onset of pancreatic tumorigenesis (Fortini 2003). The distribution pattern of oxidative DNA damage in chromatin is mainly dependent on the differences in base excision repair activity. Akatsuka et al. showed that the damage distribution differences do not take place due to OGG1 deficiency. This happens mainly due to the early recruitment of base excision repair factors to open chromatin regions after oxidative DNA damage leading toward increased 8-oxo-G accumulation in heterochromatin and gene deserts region. To repair heterochromatin efficiently, some additional DNA damage response signaling pathways like ataxia-telangiectasia mutated (ATM) signaling in double strand break repair are also required. ATM interactor (ATMIN) is another important protein in nexus with oxidative DNA damage because it is found to protect neurological tissue from oxidative DNA damage. As eukaryotic DNA is highly packaged, accessibility of the DNA repair machinery in nucleosome level is another crucial factor. This complexity of eukaryotic DNA packaging mainly the nucleosomal structure inhibits the primary steps of base excision repair. Thus, different types of chromatin remodeling proteins and enzymes are also required to remove oxidative damage efficiently (Poetsch 2020). Therefore, treating with specific drugs like trichostatin A, a deacetylase inhibitor, opens chromatin structure, thus making it accessible for DNA repair machinery and increases DNA repair efficiency. Though accessibility of the base excision repair machinery and OGG1 is the major factor for oxidative DNA damage distribution all over the chromosome, mainly in the region of tightly packed heterochromatin, but novel sequencingbased strategies helped us to investigate this arena with higher resolution. In yeast, 8-oxo-G lesions are mainly found in nucleosome-rich region of the chromatin. Even, different types of repeat elements like telomeres and microsatellites also harbor high amounts of 8-oxo-G. In humans, the guanine stretches found in the telomeric repeat sequence TTAGGG are very much prone to oxidation. Microsatellite sequences like (TGGA)n and (TG)n are also very susceptible to oxidation though they do not have guanines in stretch. As the guanine stretches in human telomeric region form G-quadruplex structure, 8-oxo-G accumulation is also found in a close nexus with DNA secondary structure formation. 8-oxo-G accumulation has a direct involvement in the folding–unfolding kinetics of G-quadruplex structure (Clark et al. 2012). Additional glycosylases like NEIL1 and NEIL3 can cover the excision of 8-oxo-G at G-quadruplex-rich regions. Impaired excision by OGG1 has also been reported in telomeric non-canonical structures. 8-oxo-G is found to destabilize G-quadruplex structures. But oxidative DNA damage can also stabilize G-quadruplex folds through conformational change by the conversion of the 8-oxo-G in a fifth guanine track into an AP site, mediated by APE1 binding. Thus, oxidative DNA damagemediated folding and unfolding of non-canonical DNA structures regulate different cellular processes and are also involved in the manifestation of several diseases (Maynard et al. 2008).

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As the DNA damage response pathways and regulations are not well understood, the effect of oxidative DNA damage in GC-rich regulatory regions like promoters, enhancers, and coding regions is tendentious. Genomewide peak-calling approaches have shown that 8-oxo-G and AP sites are found mainly at promoter regions, though this finding is contradictory because of false-positive peak-calling probability driven by the sequencing biasness of GC content in the sample. Recent findings also suggested that few specific promoters having G-quadruplex structures are the houses for 8-oxo-G. Irrespective of their position either in promoters or in any structural or regulatory part of the chromatin, G-quadruplex structures can harbor 8-oxo-G, thus establishing a correlation of 8-oxo-G accumulation with GC content. 8-oxo-G adducts are also found in open chromatin and in actively transcribed exons which are epigenetically regulated by specific histone modifications. However, at active transcription sites, single-stranded DNA can also accumulate increased oxidative DNA damage, which is repaired by transcription coupled nucleotide excision repair (TC-NER). This repair mechanism is also dependent on the accessibility of the DNA, which is mainly provided by the epigenetic modifiers. H3K36me3, an evolutionary conserved epigenetic mark for actively used exons, guides the repair mechanism for mismatch repair, which leads toward reduced rate of mutations. Till now, there is no clear understanding of the dependence of base excision repair on the same mechanism. Double strand break repair mechanism is governed through different epigenetic signals, which activate different types of chromatin remodelers, scattered throughout the genome, maintaining the architecture of the chromatin. This chromatin remodeler selectively prioritizes genes and functionally important portions of the genome. For this reason, few regions of the chromatin are highly susceptible to strand break formation, thus accumulating higher frequency of mutations (David et al. 2007). The functionally important regions of the chromatin are mainly repaired by BER machinery in different levels. At first, poly-ADP-ribose polymerase 1, CUX1 and CUX2, transcription coupled repair processes (Cockayne syndrome A and B), chromatin remodeling (CHD4), and RNA splicing machinery (SNRPF) are recruited on the sites of 8-oxo-G along with OGG1. After that, epigenetic modifiers like SET complex, histone deacetylases (HDACs), and sirtuin1 (SIRT1) are recruited on AP sites along with APE1. APE1 is also recruited on R-loops, DNA–RNA hybrids through the interaction of various factors, especially transcription factors (AP1), and splicing regulators (HNRNPL), thus regulating active transcription. Nucleophosmin (NPM1) mediates the localization of APE1. Lastly, the sequence context and the accumulation of 8-oxo-G affect the overall damage repair efficiency (Van Houten et al. 2018).

5.3.2

Nucleotide Excision Repair (NER) Pathway

Several damaging agents like UV radiation (sunrays), chemicals, or alkylating agents can induce the formation of bulky lesions on DNA. The nucleotide excision

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Fig. 5.7 A comparative representation of oxidative DNA damage repair through base excision repair (BER) and nucleotide excision repair (NER) pathways. Both BER and NER repair pathways utilize the sequence of complementary DNA strand to fix the original sequence information lost in the damaged DNA strand. Both the pathways can repair oxidative DNA lesions through several steps, i.e., recognition of the damage, excision, polymerization, and ligation, which are mediated by specific proteins and enzymes

repair (NER) pathway, which consists of more than 30 proteins, can repair a wide variety of bulky DNA lesions formed by either exogenous or endogenous agents. UV radiation (sunshine) mainly produces helix-distorting cyclobutane pyrimidine dimers (CPDs), i.e., thymine-thymine, thymine-cytosine, and cytosine-cytosine dimers and pyrimidine-(6,4)-pyrimidone products (6- 4PP). Chemicals or alkylating agents like polycyclic aromatic hydrocarbons mainly present in cigarette smoke and smoked meat can also form bulky DNA adducts. Recent research also reported the oxidative DNA damage repairing ability of NER pathway. NER is mainly consisting of two different pathways, i.e., global genome NER (GG-NER) and transcription coupled NER (TC-NER). The initiation of both the pathways is different, but they follow the same route map during elongation. GG-NER pathway first recognizes and then eliminates all the lesions, scattered throughout the genome. As the pathway involves the scanning of the entire genome, it makes it an inefficient and slow process. On the other hand, TC-NER pathway takes care of the lesions during active transcription because the lesions cause road blockages for transcription machinery. TC-NER removes all the lesions from the template DNA strand and pave the way for RNA polymerase. Both the pathways are divided into four steps: (1) recognition of DNA damage, (2) recruitment of incision complex and unwinding of DNA, (3) incision in both the DNA flanks and excision of the damaged fragment, and (4) DNA synthesis and ligation of the gaps (Kobaisi et al. 2019) (Fig. 5.7). The initial damage recognition steps of GG-NER and TC-NER are different. At first, the helical distortions and sequence alternations in DNA are recognized by the

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machinery. In GG-NER, the DNA lesions are recognized by the XPC/hHR23B/ centrin complex, UV-damaged DNA binding (UV-DDB) protein (DDB1/DDB2) complex. In the case of TC-NER, preincision complex is recruited by the XPC/hHR23B complex. The repair of UV-induced DNA lesions is mediated by the DDB2-DDB1-CUL4-RBX1 E3 ligase complex (CRL4DDB2). The XPC protein is composed of several binding domains for interacting with DNA, hHR23B, Centrin2, 8-oxo-guanine glycosylase (OGG1), and transcription factor II H (TFIIH). Thus, XPC protein is found to be associated with the repair of oxidative DNA damage. Though TFIIH is a very important transcription initiation factor, but it is an integral part of NER machinery (Lee and Kang 2019; Kobaisi et al. 2019). During active transcription, RNA polymerase II (RNA polII) gets stalled after encountering the DNA lesions, which acts as a road blockage for RNA polII. Thus, RNA polII along with TFIIH recruits all the important proteins of TC-NER to remove the lesions. Cockayne syndrome complementation group A (CSA) and B (CSB) proteins play a very critical role in TC-NER as they regulate RNA polIImediated transcription. During TC-NER, the CSB protein, a SWI/SNF ATPase, interacts with RNA polII and displace the stalled RNA polymerase from the track. The role of CSA is still not properly understood. Recent report suggests that CSA may have a possible function in TC-NER during the elongation of transcription. Damage detection and E3 ubiquitin ligase complex (CSA-DDB1-CUL4-RBX1, also known as CRL4CSA), is recruited to the DNA lesion and then promotes DNA repair, thus restarting the transcriptional process. Another crucial TC-NER factor, UV-sensitive syndrome protein A (UVSSA), has been recently discovered and characterized in detail. UVSSA interacts with RNA polII during elongation and stabilizes CSB through the recruitment of USP7, deubiquitinating enzyme to TC-NER complexes. CSB regulates the recruitment of preincision NER complex on the damaged site. With the help of CSB, CSA also recruits chromatin remodeling proteins HMGN1, XAB2, and TFIIS (Melis et al. 2013). After DNA damage recognition, GG-NER and TC-NER both intersect into the same pathway. At first, DNA damage is mainly recognized by XPC/ hHR23B complex. Then, TFIIH complex is recruited on DNA and starts the unwinding of DNA. The TFIIH complex is a crucial factor for NER pathway which is composed of 10 proteins: XPB, XPD, p8, p34, p44, p52, p62, and the CDK-activating kinase (CAK) complex: cyclin H, MAT1, and CDK7. TFIIH induces the formation of an open bubble structure in the DNA helix. Further unwinding process is accomplished by DNA helicases XPB and XPD, thus paving the way for the preincision complex to get the access on the lesions site. Some other important proteins like XPA, XPG, and RPA are also the part of the preincision complex, which are recruited on the lesions. With the help of single-stranded binding protein RPA, XPA scans the lesions and organizes the proper positioning of other repair factors on the damaged site. DNA incisions are accomplished by the endonucleases ERCC1-XPF and XPG and remove a 24–32 nucleotide single strand fragment from the damaged site. After releasing the damaged site, the gap is filled by DNA polymerases (Pol d, Pol e, and Pol j), proliferating cell nuclear antigen (PCNA), RPA, and replication factor C. At last, the nick is closed by DNA ligase (Melis et al. 2013).

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Oxidative DNA Damage Can Disrupt Cellular Function

Oxidative stress can lead toward chronic inflammation, which can form the basis of chronic diseases like cancer, cardiovascular, pulmonary diabetes, and neurodegenerative diseases (Cacciapuoti 2016). Oxidative stress can trigger a wide array of transcription factors, e.g., p53, SP1, NF-κB, HIF-1α, Nrf2, PPAR-γ, and β-catenin/ Wnt (Sharifi-Rad et al. 2020) (Fig. 5.8). More than 500 different genes including genes for growth factors, cell cycle regulatory proteins, and inflammatory cytokines are expressed through the activation of these transcription factors, thus altering the cellular equilibrium. 8-oxo-G adducts can disrupt many cellular functions through multiple mechanisms. 8-oxo-G lesions on DNA can act as road blocker, thus inhibiting the interaction between transcription factors and DNA. This disruption can either promote or impede transcription (Kong and Lin 2010; Brégeon et al. 2009). Through the recruitment of several proteins on DNA, 8-oxo-G and AP sites can also change the topology of non-canonical DNA structure like G-quadruplex, which can alter replication, transcription, gene regulation, and genome stability (Fleming et al. 2017a). The reactive aldehyde group present in the AP site reacts with the amino groups to form protein–DNA crosslinks, thus hampering the genomic integrity. G-quadruplex structure formed at the telomeric region protects the telomeric end from unwanted chromosomal fusion, thus maintaining telomeric stability. Oxidative DNA damage in telomeric regions alters the folding of G quadruplex structures, thus leading toward altered telomeric length and genomic instability (Fleming et al. 2017b). On the other hand, these oxidative lesions can also change the folding of G-quadruplex structures present in replication fork, thus

Fig. 5.8 Transcription factors which are regulated by reactive oxygen species. Peroxisome proliferator-activated receptor gamma (PPAR-γ), nuclear factor kappa B (NF-κB), activator protein 1 (AP-1), nuclear factor erythroid 2-related factor 2 (NRF2), signal transducer and activator of transcription 3 (STAT3), specificity protein 1 (SP1), tumor protein P53 (p53), and hypoxiainducible factor 1-alpha (HIF - 1α). More than 500 different genes including genes for growth factors, cell cycle regulatory proteins, and inflammatory cytokines are expressed through the activation of these transcription factors

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altering the replication. If not repaired, accumulation of 8-oxo-G and AP sites in different regions of the genome can actually alter different cellular processes like cell cycle, replication, and transcription leading toward the disruption of cellular homeostasis and manifestation of several diseases (Cadet and Davies 2017; Kobaisi et al. 2019; Boldogh et al. 2012).

5.5

Human Diseases Induced by Oxidative DNA Damage

As discussed in the previous sections, the various stressors present in the environment cause oxidative changes to the DNA that prove to be highly mutagenic. Throughout life, oxidative DNA damage and the mutations that accompany it contribute to the process of aging and the development of various age-related pathologies, such as neurodegeneration and cancer. Most of the 20,000 base lesions that are predicted to occur on average in cells under physiological settings do not advance to mutations since they are repaired or accepted during replication without causing genetic information loss or change. Unrepaired oxidative DNA damage and repair intermediates, on the other hand, pose a danger of mutagenesis. Because of the huge amounts of cancer resequencing data available for research mutagenesis and the development of novel algorithmic, statistics, and computational methodologies, the variability of mutation rates across the genome is becoming increasingly well known. 8-OH-dG lesions are well-established indicators of oxidative stress, and their potential mutagenicity in mammalian cells has led to speculation that they could be used as intermediate markers of a disease endpoint, such as cancer (Cooke et al. 2003). The fact that GC TA transversions likely originated from 8-OH-dG have been detected in vivo in the ras oncogene and the p53 tumor suppressor gene in lung and liver cancer supports this hypothesis. GCTA transversions are not specific to 8-OH-dG, although CC to TT substitutions in the absence of UV have been identified as ROS hallmark mutations in internal malignancies (Cooke et al. 2003). The accumulation of oxidative DNA damage can activate a process of permanent cell cycle arrest and trigger the formation of senescence-associated secretory phenotype (SASP) and the process of early senescence (Kumari and Jat 2021; Nelson et al. 2018). SASP factors are implicated in a variety of acute and chronic pathological processes, including cardiovascular disease (CVD), neurodegenerative disorders (NDs), acute and chronic kidney disease (CKD), macular degeneration (MD), biliary illnesses, and cancer (Pole et al. 2016) (Fig. 5.9). Obesity, diabetes, hypertension, and atherosclerosis are all linked to the inflammatory pathway mediated by IL-1, IL-6, and IL-8, as well as an increase in cellular senescence (Watanabe et al. 2017). Furthermore, SASP-driven osteoblastic trans-differentiation of senescent smooth muscle cells is associated with vascular calcification. Brain tissue biopsies indicate elevated levels of p16, MMP, and IL-6 in various neurodegenerative diseases, including Alzheimer’s disease (AD). Several harmful SASP profiles, including IL-6, IL-8, and MMP, are shared by chronic obstructive pulmonary disease, cholangitis, biliary cirrhosis, and osteoarthritis. Cancer

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Fig. 5.9 Oxidative DNA damage leads to various diseases in humans—A functional systemic regulation is directed by a tightly regulated expression of the genetic code in our cells. Therefore, DNA damage is detrimental and can cause deregulated functioning and diseased phenotype in various parts of the body

metastasis is aided by the activation of epithelial to mesenchymal transition mediated by an increase in SASP factors in response to DNA oxidative damage (Faheem et al. 2020). In summary, the oxidation-based inflammatory theory of aging (oxi-inflammaging) has been proposed, based on the close relationship between oxidative stress, inflammation, and aging: aging is a loss of homeostasis caused by chronic oxidative stress, which affects especially the regulatory systems such as the nervous, endocrine, and immune systems (Martínez de Toda et al. 2021). The immune system’s activation causes an inflammatory response, creating a vicious cycle in which

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persistent oxidative stress and inflammation feed off each other, increasing age-related morbidity and mortality (Cuollo et al. 2020).

5.5.1

Oxidative Stress in Cancer Prognosis

The lack of antioxidant enzymes in the microenvironment of tumors, as well as the excessive production of reactive oxygen species (ROS), is likely to contribute to elevated levels of DNA damage in cancer cells (Hawk et al. 2016) (Fig. 5.10). Some tumor cell lines have also been reported to produce significant amounts of H2O2 without foreign stimulation (Lisanti et al. 2011; Doskey et al. 2016), which could account for the higher levels of oxidative DNA damage seen. Increased ROS causes transcript factors and their associated genes to be constantly active, which, when combined with increased DNA damage, creates a selection pressure for a malignant phenotype present in cancer (Turgeon et al. 2018). In recent studies 8-oxo-guanine has often been used as a diagnostic marker to determine oxidative stress level and

Fig. 5.10 Accumulation of mutations, and various effects of hypoxia on carcinogenesis. Normal cells in the presence of oxidative chemical species, accumulated oxidative DNA damage like 8-oxo-Guanine formation, base adducts, etc. (fig. 5.3). These alterations lead to accumulation of mutations and genomic instability that promote the various hallmarks of cancer

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Fig. 5.11 Effects of ROS on DNA and its relation to cancer—Increase of reactive oxygen species causes various types of DNA damage leading to mutations and genomic instability that promote cancer genesis. DNA damage repair pathways and various cellular intrinsic pathways play an important role in reversing the effects of ROS on DNA damage

showed an elevated profile in various cancers and neuronal diseases (Cooke et al. 2003; Korkmaz et al. 2018). Many DNA repair processes, as well as some other cellular stress response systems including cell cycle arrest and death, are involved in determining the likelihood of genetic changes leading to cancer. The involvement of certain DNA lesions in carcinogenesis has been well documented, for example, high quantities of 8-oxo-G and other oxidative damage (Cooke et al. 2003). Furthermore, malignancies may change antioxidant and repair enzyme activity. As a result, various DNA damage repair (DDR) mechanisms may play a key role in cancer prevention. Cancer risk is a consistent pattern among people who have mutations in DNA repair genes. So, genomic instability and cancer can result from DNA damage amplified by DNA repair deficiencies (Fig. 5.11). The abnormalities found in mice knockout strains demonstrate the relevance of the BER pathway’s appropriate operation in maintaining life. Gross BER deficiencies appear to be life-threatening. XRCC1, POL, APE/HAP1, FEN1, and DNA ligase I are all embryonic fatal in mice with knockouts of their key BER proteins. However, redundancy may safeguard other steps in the BER pathway. MYH and OGG1 knockout mice have a very mild phenotype, but MYH/OGG1

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double mutant animals have a high proclivity for tumor growth (Maynard et al. 2009; Grundy and Parsons 2020; Xie et al. 2004). In 2002, a phenotype comparable to familial adenomatous polyposis was found to have mutations in the gene encoding the human MutY homolog, indicating an early relationship between a hereditary impairment in BER and cancer (Al-Tassan et al. 2002). It appears that the MutY homolog mutations observed resulted in a decreased ability to initiate the repair of 8-oxo-G•A mismatches, which likely led to an increase in G–T transversions in the adenomatous polyposis coli gene, resulting in the inactivation of the gene (Venesio et al. 2012). However, other than mutations in BER pathway genes genetic disorders can occur by mutations in other DNA repair system genes. Monoallelic pathogenic mutations in the mismatch repair genes MLH1, MSH2, MSH6, PMS2, and EPCAM linked to Lynch syndrome are also linked with a predisposition to developing various cancers (Ryan et al. 2017). POL proteins are found in 30 percent of all human cancers studied, with around half having a single amino acid alteration (Heitzer and Tomlinson 2014). Single-nucleotide polymorphism (SNP) association studies are being used to investigate more subtle correlations between cancer and other BER genes. After being subjected to environmental pollutants, people with polymorphisms in the BER genes may be more susceptible to cancer. Polymorphisms may explain why people who are exposed to the same number of carcinogens in the environment, such as cigarette smoke, have varied cancer risks. For example, most lung cancers are linked to smoking, yet only a tiny percentage of smokers will develop symptoms. A meta-analysis of the APE1 polymorphism Asp148Glu (166) found no significant link in prostate cancer, in the Asian population (Chen et al. 2016). On the other hand, the XRCC1 Arg399Gln polymorphism has been linked to specific cancers, particularly in Asian populations, but not to overall cancer risk (Xia et al. 2021; Asian Pac J Cancer Prev 2013). Individuals with the 399Gln/Gln genotype exhibited a higher risk of lung cancer among Asians (OR = 1.34, 95 percent CI = 1.16–1.54) (Cai et al. 2014), according to a meta-analysis of 17 studies examining this polymorphism and lung cancer risk (>7000 cases and >9000 controls). Zhang et al. observed a connection between the Arg399Gln polymorphism with breast cancer in Asian populations (OR = 1.6, 95 percent CI = 1.1–2.3) in a meta-analysis of four investigations (1600 cases and 1600 controls). Zhang et al. found no such link in the Caucasian population in an accompanying meta-analysis of eight studies (8000 cases and 8000 controls). Hung et al. found that individuals homozygous for the variant polymorphism (Gln/Gln) had an increased risk of tobacco-related cancers among light smokers (OR = 1.38, 95 percent CI = 0.99–1.94) but a decreased risk among heavy smokers (OR = 0.71, 95 percent CI = 0.51–0.99) in meta-analyses examining smoking associations with the XRCC1 Arg399Gln polymorphism. The gap could likely be due to distinct mechanisms impacting cells exposed to light vs heavy cigarette smoke. According to Hung et al., the XRCC1 399Gln allele has been linked to enhanced mutagen sensitivity and higher amounts of DNA adducts, which could explain why light smokers have a higher risk of tobacco-related malignancies (Hung et al. 2005).

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A fundamental feature of cancer is genome instability. For example, genomic instability in lymphoid tumors frequently corresponds to chromosomal translocations, wherein proto-oncogene loci are fused to those of antigen receptors, apparently by aberrant antigen–receptor recombination (Liu et al. 2019). MMR deficiencies also induce microsatellite instability (MIN), which increases the risk of colorectal and endometrial cancers (Li et al. 2020). Furthermore, most sporadic solid tumors have chromosomal instability (CIN) (Bakhoum and Cantley 2018). Transient CIN is thought to occur when telomeres in a developing tumor become critically short and vulnerable to chromosomal complexes, while active oncogenes and the resulting DNA-replication stress with DSB creation constantly feed CIN. Prolonged hypoxia and re-oxygenation in later stages of cancer progression may also lead to genomic instability and deregulate DDR pathways. Recently, Knijnenburg et al. report a tool for pan-cancer investigation of DNA damage repair (DDR) deficit in cancer called the Cancer Genome Atlas (TCGA) (Knijnenburg et al. 2018). Integrative genomic and molecular investigations were used to find common DDR abnormalities in 33 cancer types and correlate gene- and pathway-level modifications with genomewide measures of genome instability and reduced function. Thereby, characterization of the relevance of DDR gene function in cancer risk, progression, and therapy response could be predicted/mapped by these findings (Knijnenburg et al. 2018).

5.5.2

In Neurodegenerative Diseases

DNA repair gene mutations have also been linked to neurodegeneration. Ataxiatelangiectasia (ATM), xeroderma pigmentosum (XPA-G), Cockayne syndrome (CSA and B), and Werner syndrome (WS) are some of the most common. In addition, mental retardation is a feature of trichothiodystrophy, Bloom syndrome, and Rothmund–Thomson syndrome (McKinnon 2009). It has been suggested that Alzheimer’s disease sufferers’ neurons may have a BER genetic flaw (Weissman et al. 2007). In Alzheimer’s disease patients, elevated 8-oxo-G levels, mitochondrial deletions, and BER deficiency have all been discovered (Sliwinska et al. 2016). Because neurons have such high metabolic rates, it has been suggested that BER may play a larger role in mending DNA damage in neurons (in both nucleus and mitochondria) than other cell types. Neuronal cell death is linked to oxidative stress, which is linked to several neurodegenerative diseases. Oxidative damage is evident in the cortical region of brains with Lewy bodies which accompany dementia (Garcia-Esparcia et al. 2017), as well as in the chronic active plaques of multiple sclerosis patients (Haider et al. 2011). Furthermore, the mitochondrial version of OGG1 is upregulated in substantia nigra of Parkinson’s patients (Grünewald et al. 2019), and APE1 expression and activity are downregulated in sporadic ALS patients. APE1 missense mutations were also discovered in eight of eleven ALS cases, including sporadic and familial. However, one study found that APE1 levels were higher in ALS patients. DNA strand breaks are now commonly believed to be caused by oxidative damage, which is consistent with increased free carbonyls in the nucleus of neurons in neurodegenerative diseases. With subsequent mutations and

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dysregulation in the DNA damage repair pathways, this oxidative DNA damage is amplified to lead to further neurodegenerative progression (Coppede and Migliore 2010). Fragile X syndromes, Friedrich’s ataxia, spinocerebellar ataxias, diabetes mellitus type 2, Creutzfeldt-Jakob disease, myotonic dystrophy, and Huntington’s disease are some other neuromuscular and neurodegenerative illnesses. Similarly, mutations in the mitochondrial DNA led to impaired mitochondrial function associated with diseases like amyotrophic lateral sclerosis, mitochondrial encephalomyopathy, Leigh syndrome, myoclonic epilepsy, Leber’s hereditary optic neuropathy, and additional neuro- and myopathies.

5.5.3

In Inflammation/Infection

Numerous investigations of inflammatory diseases or infections have found higher levels of 8-OH-dG, indicating a link between inflammation and oxidative stress. The bactericidal species (O2-• and H2O2) produced by invading neutrophils, macrophages, and eosinophils harm the surrounding tissue and are a significant source of ROS. This can cause DNA damage in cells co-cultured with activated phagocytes in vitro. Inflammation can set off a chain reaction in which ROS-damaged tissues release cytokines, which encourage the infiltration of more inflammatory cells, aggravating the underlying infection. This condition could result in chronic inflammation and, as a result, persistent oxidative stress, even after the inflammatory stimulus has been eliminated. Chronic inflammation, as well as the oxidative stress it causes, has been related to the etiology of autoimmune illnesses including rheumatoid arthritis and systemic lupus erythematosus, with radical generation causing not just connective tissue damage but also cellular bimolecular alteration. DDR abnormalities can cause immunological insufficiency because genome gene mutations involving DDR factors arise during immune system development. Mutations in NHEJ factors, for example, cause B- and T-cell immune insufficiency resulting from impaired V(D)J recombination. Reactive oxygen species (ROS) are produced at a higher rate during inflammation, which then causes oxidative DNA damage. These in turn increase inflammation and DNA damage, increasing the chance of further diseases associated with oxidative DNA damage like cancer (Sahan et al. 2018). Although the precise process is unclear, observations suggest that infection-mediated inflammation may contribute to the growth of cancer by amassing potentially mutagenic DNA damage in neighboring cells. As cited by the words of Marshall and Warren (2005) from the Noble Prize press release of 2005, “Many diseases in humans such as Crohn’s disease, ulcerative colitis, rheumatoid arthritis, and atherosclerosis are due to chronic inflammation. The discovery that one of the most common diseases of mankind, peptic ulcer disease, has a microbial cause has stimulated the search for microbes as possible causes of other chronic inflammatory conditions. Even though no definite answers are at hand, recent data clearly suggest that a dysfunction in the recognition of microbial products by the human immune system can result in disease development. The discovery of Helicobacter pylori has led to an increased understanding of the connection between chronic infection, inflammation, and cancer” (Marshall and

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Warren 1984). Their words very simply explain that prolonged inflammation can be precursor to various other diseases like cancer and rheumatoid arthritis. The DNA repair mechanisms also fail due to the increasing oxidative stress and the accumulating DNA damage due to oxidative stress leads to genomic instability. The creation of reactive oxygen species (ROS) driven by inflammation, which can accelerate the rate at which genetic mutations can accumulate to cause cancer, is frequently related to the intrinsic process of genomic modifications brought on by infection (Hanahan and Weinberg 2011).

5.5.4

In Cardiovascular Diseases

The cardiovascular disease appears to have minimal links to oxidative DNA damage among the major clinical disorders. That is not to suggest that oxidative stress has no role in cardiovascular disease, which includes atherosclerosis, hypertension, heart failure, cardiomyopathy, coronary heart disease, and myocardial infarction. Indeed, there is mounting evidence indicating the role of reactive oxygen species (ROS) in the formation of atherosclerotic plaques, which, given the information presented in the previous section, is unsurprising given that atherosclerosis is widely seen as a chronic inflammatory disease (Burtenshaw et al. 2019). Whereas p53-induced cell death protects against cancer, pro-apoptotic p53 activity is deleterious in situations like stroke or heart attack. The induction of p53 by oxidative stress and other forms of DNA damage can influence the progression of atherosclerosis, implying a relationship between the DDR and cardiovascular disease (Men et al. 2021; Mak et al. 2017). Atherosclerosis also has a component that resembles carcinogenesis, particularly the initial hyperplasia of vascular smooth muscle (VSM) cells, which could be a source of DNA damage (Grootaert et al. 2018). Also, PARP (a DDR-associated protein) inhibition limits or prevents overexpression of adhesion molecules and pro-inflammatory cytokines and improves survivability in animal models of heart failure and circulatory shock (Henning et al. 2018). In a study by Kroese et al. it was seen that the presence of elevated levels of 8-oxo-guanine levels in blood and urine samples of patients correlated with a predisposition of atherosclerosis and heart failure (Kroese and Scheffer 2014). This discovery, like others, initiates the question of what biomarkers can tell us (Di Minno et al. 2016). After all, why should high amounts of 8-oxo-guanine in lymphocytes is linked to a higher risk of coronary artery disease? Do the levels of lesions in lymphocytes correspond to those in the target tissue, i.e., the heart’s blood vessels? Given the inflammatory character of atherosclerosis and the fact that lymphocytes spend the bulk of their lives in peripheral tissue rather than the systemic circulation, these cells may be exposed to the oxidizing species associated with inflammation (Senoner and Dichtl 2019).

5.5.5

Aging/Infertility and Metabolic Syndromes

As discussed earlier in this chapter, the accumulation of DNA damage slowly pushes a cell toward senescence and apoptosis. Aging is a symptom due to the accumulation

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of DNA damage due to the declining DNA repair capacity of the cell. As meiotic recombination requires the formation of DSBs, certain DDR abnormalities are likely to result in human infertility. DDR signaling is easily observable during human spermatogenesis, and a variety of hereditary DDR defects are associated with infertility or sub-fertility (Richardson et al. 2004). DDR deficiency could thus be the source of a major fraction of human infertility. Atherosclerosis, insulin resistance, and abnormal glucose metabolism are all symptoms of metabolic syndrome, which is a rather prevalent disorder. Interestingly, insulin resistance and glucose intolerance are prevalent in ATM-defective individuals, whereas animals with ATM mutations show characteristics related to metabolic syndrome and atherosclerosis. DDR-regulated kinases also target a wide range of substrates involved in glucose homeostasis as well as the insulin-AKT kinase signaling network (Schneider et al. 2006). Although there may be some indirect links between the DDR and metabolic syndrome, it is plausible that the DDR directly controls components of energy homeostasis and cardiovascular physiology that are relevant to metabolic syndrome.

5.5.6

Genetic Diseases Due to DDR Damage

DNA damage is continually present in the human genome. Inadequate protection against this damage causes genomic instability, which can lead to somatic disorders and mutations. As a result, our organism has several DNA repair processes that are highly conserved and effective. Rare disorders with DNA repair defects can occur if these repair systems are impaired due to genetic alterations in key genes. Often these genetic alterations in DDR pathway-related genes can cause inherited DDR defects which in turn cause cancers and neurodegenerative diseases (Tiwari and Wilson 3rd. 2019; Leandro et al. 2015) (Table 5.1).

5.6

Treatment of Diseases by Targeting DNA Damage and DDR Pathways

The essentiality of understanding the disease mechanisms that caused oxidative damage to nucleic acids and genetic instability is the search for probable ways to cure these diseases. While genetic therapy to complement the genes is a promising avenue of therapy for various genetic diseases, it is yet to be successfully implemented. While some progress has been achieved, such techniques have been beset by safety concerns, which have arisen primarily as a result of unwanted NHEJmediated integration of the inserted gene into tumor suppressor loci (Moehle et al. 2007). However, most prevalent cancer therapies, i.e., radiotherapy and chemotherapies, function mostly by inducing genetic damage in already genetically unstable cells speeding the process of cell senescence and apoptosis (mentioned in Table 5.2) (Helleday et al. 2008). Therefore, cancers with active DDR mechanisms cause

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Table 5.1 : Rare genetic diseases due to mutations in DNA damage repair gene Syndrome Aicardi-Goutières syndrome-1(AGS)

Mutated DDR genes TREX1, RNASEH2

DDR pathway DNA exonuclease 1

Ataxia with oculomotor apraxia type-1

APTX

NHEJ

Amyotrophic lateral sclerosis

Defective Cu-Zn superoxide dismutase (SODC, SOD1); mitochondrial DNA MRE11

Increased oxidative stress

Ataxia-telangiectasialike disorder Ataxia-telangiectasia

ATM

Microhomologymediated end joining (MMEJ) DNA ds breaks

Bloom syndrome

BLM

DNA helicase

Breast cancer type 1, early onset

BRCA1

Homologous recombination

Breast cancer type 2, early onset Cerebro-oculo-facioskeletal (COFS) syndrome

BRCA2

Homologous recombination Nucleotide excision repair (NER)

ERCC1, ERCC2, ERCC5, ERCC6

Cockayne syndrome

ERCC6, ERCC8

Down syndrome

Trisomy of chromosome 21 ERCC6L2

ERCC excision repair 6 like 2 (ERCC6L2) deficiency

Nucleotide excision repair (NER) Various pathways Nucleotide excision repair (NER)

Phenotype Severe-combined immunodeficiency, lymphomas, and hypersensitivity to ionizing radiation Ataxia, oculomotor apraxia, neurodegeneration, peripheral axonal motor and sensory neuropathy, muscle weakness The progressive degeneration of motor neurons, muscle weakness, and atrophy, leading to fatality Soft tissue sarcomas, breast cancer, brain tumors Cerebellar ataxia, telangiectasis, immune defects, predisposition to malignancy Shorter than average height, narrow face, red skin rash on sun exposure, increased risk of cancer De/demyelination, brain calcification, microcephaly, elevated CSF (cerebrospinal fluid) IFN-α, CSF lymphocytosis (CSF lymphocytes increased) Breast and ovarian cancer Degenerative disorder, craniofacial and skeletal abnormality, reduced muscle tone, impaired reflexes Abnormally small head size, stunted growth, delayed development Mental retardation, progeria Myelodysplastic syndromes, bone marrow failure syndromes, acute erythroid leukemia (AML M6) (continued)

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Table 5.1 (continued) Syndrome Familial breast cancer (non BRCA1/ 2 mediated) Fanconi anemia

Mutated DDR genes Chk2, MRN, ATM BRIP1, PALB2, FANCA-FANCL, BRCA2 (FANCD1)

DDR pathway As various checkpoint regulators Intrastrand crosslink (ICL) DNA lesions Mitochondrial oxidative pathway disorder Mismatch repair

Friedreich’s ataxia

GAA expanded repeats in frataxin (FXN)

Hereditary non-polyposis colorectal cancer (HNPCC)

MSH2, MSH3, MSH6, MLH1, PMS2, APC

Huntington disease

HTT

Scaffolds other DDR proteins

Hutchinson-Gilford progeria syndrome (HGPS) and restrictive dermopathy (RD) Inactivation of Ku70 or Ku80 in mouse models Li-Fraumeni syndrome Lig4 syndrome/ human immunodeficiency with microcephaly Nijmegen breakage syndrome (NBS)

Lamin-A

Double strand break repair, homologous recombination

Ku70 Ku80

Nonhomologous end joining (NHEJ) Cell cycle halting Nonhomologous end joining (NHEJ)

NBS-like syndrome

p53 DNA ligase IV, XLF/Cernunnos

NBS1

RAD50

Recruitment of DNA phosphokinase to damage repair sites Double-stranded break, DNA recombination, telomere integrity

Phenotype Predisposition to medium/ late-onset breast cancer Ataxia, neurodegeneration, oculomotor apraxia, and peripheral neuropathy Limb ataxia, cerebellar dysarthria, sensory loss, skeletal deformities Congenital abnormalities, progressive bone marrow failure, prone to AML, squamous carcinomas of head, neck, or gynecological system Progressive chorea and dementia, severe neuronal loss in the striatum and cerebral cortex Accelerated aging (HGPS); neonatal lethality (RD)

Premature aging, cancer predisposition, lymphomas Colon and gynecologic cancers Microcephaly, leukemia, immunodeficiency, and developmental and growth delay Microcephaly, growth retardation, mental retardation, immunodeficiency, cancer predisposition NBS-like phenotype

(continued)

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Table 5.1 (continued) Syndrome Parkinson’s disease

Mutated DDR genes Mutations in α-Synuclein and Parkin variants

DDR pathway Double-stranded breaks repair pathway

Primary microcephaly 1

MCPH1/BRIT1

RIDDLE syndrome

RNF168 (RIDDLIN)

Double-stranded breaks repair pathway Chromatin modifier

Rothmund–Thomson syndrome

RECQL4, ANAPC1

DNA helicase

Seckel syndrome

ATR, CEP152, CEP63, NINI, RBBP8. ATRIP, ATR

DNA crosslinkage

Spinocerebella ataxia with axonal neuropathy (SCAN1)

TDP1

DNA topoisomerase

Trichothiodystrophy

ERCC2, ERCC3, GTF2H5

Nucleotide excision repair (NER)

Triple-A syndrome

Mutation in AAAS Gene

Werner syndrome

WRN

DNA damage response impairment and defective DNA repair DNA helicase

Xeroderma pigmentosum

XPA-G, XPC, ERCC2, pol H

Base excision repair

Phenotype Tremor, bradykinesia, posture rigidity and postural instability, degeneration of dopaminergic neurons in the substantia nigra area Microcephaly, mental retardation Radiosensitivity, immunodeficiency, dysmorphic features, and learning difficulties Red blistering rash on the face, thinning skin, stunted growth Marked microcephaly, primordial dwarfism, dysmorphic facial features and mental retardation, possibly AML Breast and ovarian cancer; predisposition to pancreatic, prostate, and gastric cancer and melanoma Sensitivity to UV, short stature, intellectual disability, respiratory infection prone, risk of skin cancer Adrenal insufficiency, achalasia, alacrima, neurodegeneration, autonomic dysfunction Abnormally slow growth rate, alopecia, canities Skin pigmentation deficient, skin cancer, neurodegeneration, ocular surface abnormalities, acute sunburn

Additional information on human diseases with DDR defects and nuclear or mitochondrial DNA instability can be found at: http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=gnd and https:// mseqdr.org/mitobox.php, http://repairtoire.genesilico.pl/proteins/diseases/ (Milanowska et al. 2011; Jackson and Bartek 2009; Shen et al. 2016)

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Table 5.2 Various types of cancer treatment targeting increase in genomic instability via oxidative damage to DNA, or by inhibition of wild-type DNA damage repair pathways Cancer treatment Radiotherapy and radiomimetics Ionizing radiation Monofunctional alkylators Alkylsulphonates Nitrosourea compounds Temozolomide nitrogen mustard Mitomycin C Cisplatin Cytotoxic antimetabolites 6-mercaptopurine fludarabine 5-fluorouracil gemcitabine cytarabine pemetrexed methotrexate Replication inhibitors Purine-like: Cladribine (2-chlorodeoxyadenosine), Clofarabine [2-chloro-9-(2′deoxy-2′ fluoroarabinofuranosyl)adenine], Fludarabine (9-β-D-arabinoside-2-fluoroadenine) Pentostatin (2′-deoxycoformycin) Pyrimidine-like: cytarabine [1-β-D-arabinofuranosylcytosine (Ara-C)] gemcitabine [2′,2′-difluorodeoxycytidine (dFdC)] 5-aza-deoxycytidine tezacitabine Topoisomerase inhibitors Camptothecins (topo I) etoposide doxorubicin, Epirubicin, Daunorubicin (topo II)

Types of DNA lesions induced Single strand breaks, double strand breaks, base damage Base damage, replication lesions, bulky DNA adducts Double strand breaks, DNA crosslinks Cytotoxic metabolite inhibits base pairing leading to base damage and replication lesions

Double strand breaks, replication lesions, nucleic acid homologues

Double strand breaks, single strand breaks, replication lesions, anthracyclines also generate free radicals

cancer therapy resistances and similar efficacy of treatment cannot be achieved in different types of cancer. DDR inhibition has thus been proposed as a way to improve the efficacy of radiation and DNA-damaging chemotherapies, and several DDR-inhibitory medicines are currently under pre-clinical and clinical investigation to explore this hypothesis (Table 5.2) (Cheng et al. 2022; Berdis 2017). Due to selection factors working during tumor evolution, many cancer cells lack one or more parts of the DDR. The presence or absence of DDR components is frequently associated with a favorable treatment outcome. Because several DNA-repair mechanisms have similar functions and because one process can occasionally “back up” for deficiencies in another, inhibiting pathways found in cancer cells should have a bigger impact on cancer than normal tissues in some circumstances. A good example is drugs that act as an inhibitor to the enzyme PARP-1, which binds SSBs and BER intermediates to promote these repair processes. PARP inhibitors, for example, are relatively innocuous to normal cells but extremely hazardous to HR-defective cells, particularly those with BRCA1 or BRCA2 mutations (Bryant et al. 2005) (Fig. 5.12). PARP-1 inhibitors are currently

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Fig. 5.12 PARP inhibition and its effects on downstream signaling. PARP-1 binds single-stranded or double-stranded DNA and promotes BER repair pathway. PARP inhibitors inhibit binding to DNA and prevent the BER mode of repair of DNA. In such a case, normal cells can repair the break by homologous recombination. However, in cancer cells often homologous recombination pathways are deficit; therefore, it can result in selective cell death of HR/BRCA-deficient cancer cells

being tested in BRCA-defective cancer patients in Phase 2 trials based on promising Phase 1 evidence (Shah et al. 2021). Similarly, CHK1 inhibition is said to make p53-deficient cells more vulnerable to DNA-damaging chemicals than p53-proficient ones (Del Nagro et al. 2014). Therefore, the advent of diagnostic techniques to distinguish DDR variations among tumor and normal cells offers a lot of potential for intelligently personalizing DNA-damaging and DDR-inhibitor therapy for each patient. Furthermore, because DDR activation is common during cancer development, screening for DDR markers might improve cancer detection’s realism and sensitivity, as well as allow for early diagnosis of pre-malignant disease (Nikolaishvilli-Feinberg et al. 2014). Not just cancer we saw in this chapter the role of over-activation of PARP-I on ischemia-reperfusion episodes, which might be targeted by PARP inhibitors for modulation (Pacher and Szabó 2007). Inhibition of PARP-1 also protects against traumatic brain injury, endotoxic shock, chronic inflammation-induced tissue damage, and drug-induced diabetes as tested in some animal models. Likewise, p53 dysfunction is a common cause of inflammatory and cardiovascular diseases so pharmacological modulation of p53 or its upstream activator ATM could mitigate such pathologies (Moroni 2008; Guevara et al. 1999; Thrasher et al. 2017). Furthermore, with our growing understanding of how DNA damage and DDR-events are linked to neurological disease and other age-related degenerative diseases, it is tempting to believe that DDR-modular medications (Table 5.3) (Cheng et al. 2022) will one day be utilized to reduce or prevent such conditions and potentially even some components of the normal aging process.

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Table 5.3 Drugs in clinical trial against various prominent DDR-associated genes (Cheng et al. 2022)

5.7

Drug Olaparib, Talazoparib BAY-1895344 AZD0156 AZD7648 AZD1775 OCT245737

Inhibition on DDR gene PARP ATR ATM DNA-PK WEE1 kinase CHK1

Conclusion

In summary, there are diversified repair pathways and different classes of proteins and enzymes that repair oxidative DNA damage in different level of the chromatin with specificity. The detailed mechanism and the consequence in each level are not properly understood due to lack of technology. Overall, the fidelity of oxidative DNA damage repair is completely dependent on the cumulative effect of all the factors in a specific cell. Unrepaired oxidative damage causes dysfunction and detrimental effects to our system, leading toward several diseases. The accumulation of common oxidative products like 8-oxo-G and 8-oxodG in DNA level can be utilized to evaluate the degree of oxidative damage in our cells. The concentration of these oxidative products in our body fluids can also impart valuable insights regarding disease onset, progression of the disease, therapy effects, and the risk assessment. As we already documented that these oxidative DNA adducts are found at an increased rate in different cancers, cardiovascular diseases, neurodegenerative diseases, and other chronic diseases. But it is very difficult to diagnose these diseases only based on one biomarker. To increase the diagnosis sensitivity of these diseases, we need to use combination of multiple markers followed by high-resolution imaging and early testing. To achieve this, we need to develop new-age technology and methodology to unravel all the important levels of DNA damage repair mechanism in high resolution. Then only, we can be able to use this oxidative damage as disease biomarkers in years to come. Oxidative DNA adducts can provide us new insights and pave the way for a new therapeutic window of drug development and diagnosis of the oxidative stress-mediated diseases. The integration of oxidative damage response and repair pathways is not well understood, thus needing deep exploration. To date, the clinical applications of 8-oxo-G/8-oxo-dG are in the juvenile stage due to lack of technology and research. Thus, in years to come, we have to conduct large-scale research to unravel the nexus between the formation and repair of oxidative DNA adducts and their role in disease development and diagnosis.

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Nucleic Acid in Nanotechnology Debopriya Bose, Laboni Roy, Ananya Roy, and Subhrangsu Chatterjee

6.1

Introduction

Nucleic acids and proteins possess intrinsic nanoscale level features that may be utilized as powerful building blocks in designing and fabrication of nanostructure and nanodevices. Nucleic acid (NA) nanotechnology is referred to as the engineering of the programmable molecular recognition properties of natural and synthetic nucleic acids for assembling structures with nanometer scale precision (Michelotti et al. 2012). Since the identification of the double-helical DNA structure, the story of nanotechnology began (Hunter 2018). DNA is described as the building block of life because of its role in storage and processing of information and transferring it from one generation to the next generation. DNA comprises nucleobases, A, G, C, and T, while RNA is a polymer containing nucleotides A, G, C, and U. The complementary base pairing feature between A and T, A and U, and G and C via hydrogen bonds and self-assembling capability have enabled programmable self-assembly and structural prediction-based synthesis of nucleic acids which in turn has opened new avenues for application of nucleic acids beyond their original function of storage and transmission of genetic information (Jin et al. 2020). The pioneering work of Robert Letsinger and Marvin Caruthers enabled the synthesis of virtually any imaginable nucleic acid sequence (Krishnan and Seeman 2019). DNA nanotechnology combines and assembles motifs formed by nucleic acid strands to form ordered 2D and 3D lattices which serves as regulatory templates for assembly of foreign material/analyte (Jin et al. 2020). Nucleic acid chemistry plays a vital role in the success of DNA nanotechnology. The residue level addressability of DNA polymer is one of its great advantages to be used in DNA nanotechnology as it allows the positioning of functional groups on DNA (Krishnan and Seeman 2019; Madsen and

D. Bose · L. Roy · A. Roy · S. Chatterjee (✉) Department of Biophysics, Bose Institute, Kolkata, West Bengal, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 S. Chatterjee, S. Chattopadhyay (eds.), Nucleic Acid Biology and its Application in Human Diseases, https://doi.org/10.1007/978-981-19-8520-1_6

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Gothelf 2019). The characteristics of DNA that makes it perfect choice for programmed self-assembly are efficient digital encodability of Watson–Crick base pairing, DNA hybridization properties, the ease of oligonucleotide synthesis of any sequence through automated synthesis, and high stability and inertness of the molecule (Madsen and Gothelf 2019). The highly precise assembly process of DNA and RNA nucleotides is utilized to generate molecular scaffolds, loops, junctions, and hairpins with potential for building nanostructures for nanoscale applications (Hunter 2018). Non-canonical DNA structures like GQ and iM have been used as building blocks to design chemically responsive DNA-based molecular switches (Krishnan and Seeman 2019). Attempts for harnessing the properties of nucleic acid for crafting artificial structures in nanotechnology started in the early 1980s; Ned Seeman’s pioneering work on artificial DNA scaffolds was the first imprint in the field of nucleic acid nanotechnology (Madsen and Gothelf 2019). In the early 1980s, DNA branched junctions were selected as basic building blocks for construction of 3D periodic networks (Hunter 2018). The actual origin of DNA nanotechnology happened in 1982 when Nadrian Seeman proposed the use of DNA to construct a 3D periodic network. And in 1983 he successfully synthesized four-arm junction structure of DNA. The first higher order geometrically complex 3D DNA lattice with six-arm junctions was also discovered by Seeman (Hunter 2018). This was followed by the conceptual foundation/origin of NA nanotechnology which is the discovery of Holliday junction which comprises four double-stranded arms joined together around a central point (Jin et al. 2020). Later in another study a stiffer and more stable geometric structure was found compared to the Holliday junction, which is a double-crossover (DX) molecule that comprises two double helices aligned parallelly with strands crossing between them. 2D and 3D DNA structures were formed using DNA tiles, DX molecules, and additional junctions (Jin et al. 2020). A DX structure can be expanded into a higher order DNA motif which can be assembled into complex 2D array, for example, 4x4 DNA tile that self-assembles to form uniform-width nanoribbons, 2D nano-grids, and ladder-like grids (Madsen and Gothelf 2019). Strand displacement underwent a massive growth helping in designing of a plethora of biosensors and their use in construction of molecular robots along with diverse nanodevices (Krishnan and Seeman 2019). Thus, this field got more attention in the late 1990s with the discovery of DNA strand displacement reactions and DNA tiles, and the invention of DNA origami by Paul Rothemund in 2006 accelerated the interest of fascinated researchers to focus on the progressive expansion and application of nucleic acid nanotechnology in biomedicine, optics, biotechnology, and nano-fabrication (Jin et al. 2020; Madsen and Gothelf 2019). His invented DNA origami structure comprised a single-stranded DNA (staple) that can produce different robust and precise structures such as polyhedrons, curvatures, meshes, nanorobots, nanochannels, and wireframes (Jin et al. 2020). DNA origami provides a powerful treatment platform because of its structural diversity, and by combining multiple DNA origami structures, DNA devices free of size limitations can also be constructed. DNA origami and DNA brick system democratized the

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architectural construction of DNA nanostructure on a multi-nanometer scale (Krishnan and Seeman 2019; Madsen and Gothelf 2019). DNA nanotechnology has been well established. The pioneering work of construction of RNA nanoparticle through the process of self-assembly of several re-engineered natural RNA molecules was reported in 1998 (Guo 2010). The potential of nanomedical applications of RNA nanotechnology in cancer treatment, genetic disease diagnosis, viral infection diagnosis, and treatment has enticed the interest of researchers to focus on the growth of RNA nanotechnology (Guo 2010). RNA can be designed and manipulated utilizing the flexibility in structure and diversity in function. RNA strands can form a large variety of stem-loop structures via inter- and intra-molecular interactions, which is utilized in making “dovetail” joints between building blocks (Guo 2010). The non-canonical base pairing between G with A or U along with the intrinsic capability of RNA to form loops and motifs allows researchers to design complex secondary structures such as aptamers, ribozymes, and siRNAs with special functions. In RNA the 2′-OH in ribose locks it into a 3′-endo chair conformation that favours A type configuration in RNA. The base stacking governed by van der waals interaction with the sum over numerous base pairs adds to the helical stability6. RNA–RNA double helix possessing lower free energy (-G0 helix) than DNA is more stable than DNA double helix (Guo 2010). The versatility of RNA structure, low free energy, structural controllability in construction of dimers, trimers, or polygons, self-assembling property, and biocompatibility make RNA ideal for application in nanotechnology (Guo 2010). There remains abundant prospect of innovation with huge potential of nucleic acids for a multitude of applications as nanodevices and nanomaterials.

6.2

Structural DNA-Based Nanotechnologies

The frequent use of nucleic acids (especially DNA) in the design of an abundance of nanostructures is attributed to its chemistry. The bonding patterns of nucleic acids by Watson–Crick pairing are immensely predictable, and this allows researchers to program the formation of both 2D and 3D assemblies made from nucleic acids. Additionally, the properties of the DNA helix are extremely well known, and the hydrophobic interior of the double helix leaves minimal space for secondary interactions. Nucleic acids are also immensely polymorphic and form reliably stable structures. These factors along with their ease of synthesis and low cost have led to very fast development in this field. As research in the field continues to progress, scientists have made use of high-throughput tools to synthesize more and more complex DNA and RNA nanostructures. These structures along with serving as inert materials can also be functionalized by chemical modification of the nucleic acids. The field of structural DNA nanotechnology relies on the ability of DNA strands to form branch motifs or junctions which can further be assembled via sticky ends to generate higher order structures. An example of a branch junction naturally formed by DNA is the Holliday junction. These structures are generally unstable due to their inherent sequence symmetry, but a stable form of this structure can be designed by

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Fig. 6.1 DNA nanomotifs. (a) An immobile Holliday junction. (b) A DX molecule containing two 4-arm junctions assembled via two crossovers into one structural unit. (c) A 4X4 tile generated by linking four immobile Holliday junctions using a central strand

limiting this symmetry. Indeed, Seeman designed such an immobile 4-arm junction (Fig. 6.1a) and further used the branching power of DNA to develop the 5-, 6-, 8-, and 12-arm junctions (Wang and Seeman 2007). These junctions were highly flexible and conformationally diverse, thus prompting the development of the double-crossover (DX) molecule (Kolpashchikov et al. 2011). The DX molecule had two 4-arm junctions assembled via two crossovers into one structural unit (Fig. 6.1b). This generated a rigid molecule with limited conformational dynamics, and it was used to synthesize a host of higher order DNA nanostructures. From this simple DX tile, the 4X4 tile was generated by linking four immobile Holliday junctions using a central strand (Fig. 6.1c). The tile so formed has a fourfold

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symmetry, and T4 loops placed at four corners of this strand limit stacking interactions between adjacent arms. With this basic structural motif in mind, we discuss the development of 2D or 3D DNA lattices.

6.2.1

DNA Tiles

DNA tiles can simply be regarded as the building blocks for higher order DNA nanostructures. These tiles can be hierarchically arranged to form complex designs through self-assembly in a method similar to the use of planks to construct complex architectures. The lattices derived from these tiles are connected by “nails.” The lattice so formed is stable if the tiles are more rigid compared to the nails connecting them. DNA tiles can be constructed by using either small DNA motifs or DNA origami units. DNA origami structures are discussed in the subsequent sections, and these structures can be used as repeating units or tiles for assembling larger and more complex structures. Small DNA motifs are generally used as tiles in the fabrication of small DNA origami units. These contain an array of short oligonucleotides which add up to about 100–350 bps per unit. The largest DNA motifs have a size around ten nanometers and are exemplified by the DX and 4X4 tile mentioned above. The assembling of such tiles into higher order structures was initially achieved by the use of sticky ends which could act as nails (Fig. 6.2a). Each DNA tile is constructed from an array of connected DNA helices, and to join multiple tiles, some of these helices are embedded with sticky ends. This means that these helices have an unpaired overhang which is complementary to the overhangs on an adjacent tile. Later blunt

Fig. 6.2 Assembling DNA tiles and bricks. (a) Four immobile junctions joined via sticky ends to form a lattice. (b) Joining of DNA helices via sticky ends, blunt ends, or a combination of both. (c) DNA bricks represented as Legos and joined via complementary sequences

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ended DNA helices were also joined by the use of stacking interactions between adjacent helices. Further a combination of sticky and blunt ends can also be used to fine-tune the joining process (Fig. 6.2b). Non-Watson–Crick base pairings are also sometimes utilized to make the assembly more thermodynamically stable. DNA tiles have been used extensively in the modelling of complex higher order structures, but the small size of the DNA motifs may sometimes pose a problem. Additionally, the DNA strands used have to be mixed in precise stoichiometries and must be highly pure to avoid the formation of erroneous structures. The use of DNA motifs thus often leads to lower yields of the final structure. In this regard, the use of DNA origami units as tiles has become increasingly popular (Parikka et al. 2021).

6.2.2

DNA Bricks

When the concept of DNA tiles was extrapolated to a 3D domain, the resulting structure behaved as a DNA brick. The earliest DNA bricks were designed to be made of 32 nucleotide strands that can be visualized as four domains of eight nucleotide each. These four domains contain two head domains and two tail domains. The head domains are the regions adjacent to the phosphate linkage, and the two regions adjacent to the head domain are the tail domains. The structure of the brick can also be explained as two 16 bp antiparallel double helices that are connected via a phosphate linkage at the junction of the two head domains. This brick unit can interact with another brick having partially complementary sequences. Upon interaction one DNA brick lies perpendicular relative to the other brick and three parallel DNA helices are obtained. This 90° helical twist is observed as a result of the 3/4th right-handed helical twist of the 8 base pairs of DNA that are in the complementary positions between the two bricks. The assembly of DNA bricks to form large and complex structures can be understood by conceptualizing each brick as a piece of Lego. Each Lego brick in this design has two arms and two binding points and any two pieces always interact in a perpendicular orientation (Fig. 6.2c). Just as Legos can be assembled to form highly complex structures, DNA bricks can also be attached by altering the sequence of each brick to maintain appropriate complementarity to its neighbouring brick to obtain highly complex target structures (Ke et al. 2012a).

6.2.3

DNA Origami

The field of DNA nanotechnology has progressed immensely with the development of the DNA origami technique. In this technique, researchers try to visualize target shapes as an assembly of DNA molecules. For this purpose, they also make use of various 3D modelling softwares and render target shapes as outline meshes composed of DNA helices. DNA origami involves the use of a large number of short oligonucleotide strands (known as staple strands) to correctly fold a long DNA strand into specific designs. In 2003, Reif, Yan, LaBean, and colleagues generated

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complicated DNA lattices using a long scaffold strand which could form a barcode lattice by interacting with short ssDNA strands (Yan et al. 2003) (Fig. 6.4a). This fundamental design later led to the development of DNA origami by Paul Rothemund in 2006 (Rothemund 2006) (Fig. 6.3). The most common DNA strand used as the scaffold is the genome of the M13mp18 virus. Since their discovery this field has progressed enormously, and now highly precise softwares are available to facilitate the design of any structure in the nanoscale using DNA origami. DNA origami can be used to form both single-layered and multi-layered structures. The steps of development of a DNA origami structure are now well defined: • Choose a desired shape • Create the basic scaffold-staple layout and use software programs to calculate the sequence of the scaffold and each staple strand • Prepare each oligonucleotide strand as found from the software • Anneal the DNA sequences in appropriate buffer and temperature conditions • Add functional moieties if necessary • Visualize the final structure and purify it if necessary Recently, methods have been developed to cross-link DNA origami structures without the need for specific temperature ramps, using only triggers such as light or various chemicals. Single-Layered DNA Nanostructures Single-layer DNA origamis can be broadly subdivided into tightly packed and wireframe designs. The first study of DNA origami reported by Paul Rothemund describes a tightly packed design. In this study, he reported the design of several planar structures including simple structures like rectangle, triangle, and five-point shapes. Additionally, he also designed nanometer-sized smiley faces using this technique. To design these structures, a desired planar structure was generated by a layer of DNA cylinders. Each cylinder represents a DNA double helix. These helices were joined by introducing crossovers between adjacent helices. This caused one strand to remain continuous and serve as the scaffold. The shorter staple strands are all complementary to parts of the scaffold and can travel between adjacent helices through the crossover points (Fig. 6.4b). Each staple strand is about 15-60 nt long, and their sequences are determined by the routing pattern of the scaffold strand, the positions of the crossovers, and the nick point positions. To finally generate the desired shape, all the DNA strands are slowly annealed in a one-pot reaction (Rothemund 2006). Later this method was utilized to develop more complex structures such as China’s map (Qian et al. 2006) and a depiction of dolphins (Andersen et al. 2008). Curved structures were generated by increasing the distance between crossovers at the outer helices. This results in the development of a tension causing the planar structure to bend at the outer edges. Apart from 2D structures, 3D structures have also been developed from planar surfaces. For this purpose, two strategies have been implemented. In the first technique, a 2D planar

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Fig. 6.3 Developing shapes with DNA origami. (a) Diagram illustrating the DNA origami design. (b) 2D or 3D DNA origami shapes—(i) rectangle, (ii) triangle with rectangular domains, (iii) Star, (iv) smiley

Fig. 6.4 Development of higher order structures using DNA. (a) A DNA barcode lattice where hairpin formation is assigned a value of 1. Stacks of the lattice when assembled can represent a barcode lattice. The black dots in the lattice are the hairpins. (b) Development of a 2D structure using DNA origami. The scaffold strand is red and the staple strands are orange, green, black, and blue

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Fig. 6.5 Generation of 3D structures using DNA origami. (A) 2D shapes with a partially unpaired scaffold can be associated via hinges to develop 3D structures such as a box with a controllable lid. (B) Generation of non-planar curved structures using DNA origami. In these structures, the distance between adjacent crossovers is highest in the outermost layer and lowest in the innermost layer. This generates a tension which makes the nanostructure non-planar and curved allowing the development of complex structures. Distance between adjacent crossovers is the same in each layer of a planar structure

structure is formed as discussed above. But the scaffold strand in this planar surface is not completely base-paired by staple strands. So the rigid 2D structure can be dissected into distinctly shaped segments. The individual 2D shapes are then associated with other such shapes using hinges, thus allowing the formation of a 3D structure. This strategy has been successfully used to develop boxes with controllable lids (Andersen et al. 2009) (Fig. 6.5A) and other structures like a DNA prism (Endo et al. 2009). The alternative strategy for the development of 3D DNA origami structures uses plane linkages. Crossovers are introduced into this structure having a distance that differs from an integer number of half turns. This creates a situation where the DNA helices are unable to form a planar structure (Fig. 6.5B). This strategy was further combined with the in-plane curving technique mentioned earlier to generate highly complex 3D structures such as a 3D sphere, a football, and a nano-sized flask (Han et al. 2011). Another category of single-layer DNA origami structures are the wireframe structures. The wireframe DNA structures were first developed by Han et al. (2013). Just as the basic unit of the tightly packed structures discussed so far is the DX tile, the basic unit of the wireframe structure is the gridiron. The gridiron unit contains four 4-arm junctions that are connected to develop a square frame. Many

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Fig. 6.6 Development of wireframe-based structures. (a) A gridiron unit containing four 4-arm junctions connected to develop a square frame. This basic unit can be assembled to generate lattices. (b) Complex wireframe structures developed by using multi-arm junctions. These structures may have a single scaffold strand or multiple scaffold strands. (c) Rendering a target structure into an array of triangular meshes using a DNA helix

such units are connected by a scaffold strand to generate 2D lattices which can either be planar or be curved (Fig. 6.6a). The process of curving the planar structure is the same as for the tightly packed system. Another method to develop more complex wireframe structures was later developed by using a number of multi-arm junctions. As discussed above, T4 loops placed at four corners of this strand limit stacking interactions between adjacent arms. Here by controlling the length of the Tn loops at each vertex and the number of unpaired bases in the scaffold strand, the angle formed between each arm can be tuned. Complex shapes can be designed by connecting such differently angled multi-arm junctions (Zhang et al. 2015) (Fig. 6.6b). Each edge of the lattice contains two double helices in this structure, and connection between each unit is made by the scaffold strand which traverses each arm twice to generate the target design. Scaffold-free approaches have also been developed to design wireframe structures. This system is much more generalizable, and any DNA building block such as single strands, multi-arm junctions, or DX tiles can be used to build the nanostructure (Piskunen et al. 2020). To facilitate the design of wireframe origami structures, a design algorithm called DAEDALUS (DNA Origami Sequence Design Algorithm for User-defined Structures) was created in 2016. DAEDALUS can analyse any 3D solid object and give the user a list of the DNA sequences and multi-arm junctions that are needed to synthesize this object in nanometer scale (Veneziano et al. 2016). Alternatively, Benson et al. used a new approach to construct wireframe structures. In their design, the target structure was divided into an array of triangular meshes constructed using DNA helices. In this system, most of the edges contained only a single DNA helix so that the scaffold strand

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traversed most edges only once to form the final structure (Benson et al. 2015) (Fig. 6.5c). Since their initial discovery more and more complex single-layered DNA origami structures have been designed by scientists. Although these structures are highly complex and compact, their applications often remain limited. This is because their flexibility and heterogeneity make them unusable in very precise operations. Additionally, it has also been observed that the scaffold DNA sometimes opens and closes at random making the structure somewhat unstable. Multi-Layered DNA Nanostructures These structures contain multiple layers of the single-layered structures and are more widely applicable as this stacking significantly reduces flexibility. The first report of such a structure came in 2009 in a study by Douglas et al. In this study, the authors used a honeycomb pleat-based strategy to develop a range of 3D structures. The honeycomb strategy can be explained by visualizing the scaffold strand as an array of antiparallel double helices that has been laid down. In this structure, the angle of attachment between two neighbouring helices is ±120°. The helices are connected by crossovers, and each helix has three neighbours. The helices formed in this structure are given initial geometric parameters such that there are 21 base pairs for every two turns of the helix. The crossovers in this structure are placed at regular intervals of 7 base-pairs. The repeating unit in this structure is a six-helix bundle, and each bundle is connected by the scaffold strand. When this structure is packed into stacks, the cross section takes the shape of a honeycomb lattice and this design strategy was used to build multiple 3D shapes such as monolith, square nut, railed bridge, genie bottle, stacked cross, and a slotted cross (Douglas et al. 2009) (Fig. 6.7a). Later more compact lattice designs were demonstrated in other studies. For example, a square lattice was demonstrated in which the basic unit is a four-helix bundle where the relative orientation between neighbouring helices (according to scaffold route) is 90° and the crossovers are placed at 8 bps apart (Ke et al. 2009) (Fig. 6.6b). Another lattice arrangement with a higher packing density has been designed. In this design, each DNA helix has six neighbouring helices and the basic repeating unit can be defined as a three-helix bundle in a triangular lattice. The adjacent helices are linked at a 60° angle by crossovers placed either 9 or 13 bps apart (Fig. 6.7b). These three lattice designs can also be combined in a single structure to develop more complicated arrangements (Ke et al. 2012b). Curves and twists were introduced into multi-layered DNA origami structures by two methods. In the first method, bends could be created having an angle between 30° and 180°. Bends in this method were created by introducing insertions or deletions at junction points and by adjusting the position of the crossovers. When the regular spacing of the crossovers is altered, a tension builds in the helices leading to curving of the structure. Insertions and deletions at specific points introduced curvature radii that could range from 6 to 64 nm, thereby allowing high tunability to the structure (Dietz et al. 2009). The second strategy to create curved designs is based on the principle of tensegrity. The principle of tensegrity suggests that when isolated components are under compressional forces within a system that has a continued tension, the isolated components

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Fig. 6.7 Multi-layered DNA origami nanostructures with different lattice designs. (a) Structure formed by a lattice containing a six-helix bundle as the repeating unit. The cross section of such a structure is a honeycomb lattice. (b) More compact lattice designs for development of multi-layered structures. The square lattice contains a four-helix bindle as the repeating unit. A yet higher packing density is achieved when the repeating unit is a three-helix bundle in a triangular lattice. (c) Woven lattice-based 3D architecture

show a vigorous strength-to-weight ratio, thus forming highly resilient units. In DNA nanostructures tensegrity was achieved by combining the rigidity of multilayered designs with the flexibility of single-stranded DNA units. This approach was successfully applied in the design of a DNA prism containing three multi-layered DNA bundles and nine ssDNA units (Liedl et al. 2010). The bundles created the compression components and the ssDNA regions act as tensed cables. A novel method to develop DNA origami structures was reported by Hong et al. Instead of arranging DNA helix bundles in a parallel arrangement, this approach makes use of crossover motifs to generate a woven lattice like 3D architecture (Hong et al. 2016). The scaffold strand in this structure travels between each layer and maintains the orientation of the helices in each layer (Fig. 6.7c). Multi-layered 3D origami structures require a higher folding time and high concentrations of Mg2+ ions during folding. Mg2+ ions minimize the electrostatic repulsions between the closely packed DNA helices. Even so, the yield of the desired multi-layered structure is often lower

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compared to the single-layered DNA nanostructures. Scientists have tried to circumvent the problem of low yield by a highly precise design of the scaffold route and by introducing staple breaks. The DNA origami structures discussed thus far can be further scaled up for the design of increasingly complex structures. For this purpose, the length of the scaffold strand can be increased by processes such as polymerase chain reaction (PCR) and rolling circle amplification (RCA). Alternatively, a small DNA origami unit may be used as a basic repeating unit for the construction of larger structures.

6.2.4

DNA Crystals

Researchers have tried to develop DNA crystals from a range of basic DNA motifs, but the first successful motif that could be used to form a DNA crystal is the tensegrity triangle. The tensegrity triangle is a rigid structure composed of seven strands. Three strands that form the edges of the triangle, three other strands present at the vertices that form crossovers, and a central nicked strand that is partially complementary to the three edge strands complete the double helices and crossovers. The double helices connected through the four-arm junctions make the alternating structure necessary for a tensegrity structure. These triangles can be connected to each other via sticky ends placed at the corner positions of the triangle. Each corner has two arms containing sticky ends, so a single triangle can make connections to six helices to form a 3D crystal (Zheng et al. 2009) (Fig. 6.8A). Later related structures such as a tensegrity square and tensegrity hexagon were also used to develop differently shaped crystals by using the concept of sticky ends (Simmons et al. 2016). While 3D crystals were generated from these structures by a hierarchical assembly of preformed 2D lattices, DNA bricks were used to develop 3D crystals directly via addition of individual bricks. As mentioned before DNA bricks can be idealized as Legos and two bricks are connected through maintaining a complementary surface. This means that the bottom layer of one brick has strands complementary to the top layer of the other (Ke et al. 2014). With the development of DNA origami, it also became possible to use DNA origami structures as building blocks for the development of crystals where the final structure was determined by the structure of the building block (Zhang et al. 2018). Recently, robust tensegrity triangle crystals have been developed that can withstand temperatures up to 65 °C by using DNA ligase-mediated post-assembly ligation to connect the sticky ends via covalent bonds (Li et al. 2019).

6.2.5

DNA Nanotubes

Nanotubes are ubiquitous in biological systems and function as membrane channels, cytoskeletal filaments, and tunnelling nanotubes. Biological nanotubes are key elements maintaining cellular structure, regulating cellular transport, and transmitting information. The development of synthetic biology has prompted the

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Fig. 6.8 Formation of DNA crystals, nanotubes, and hydrogels. (A) Lattice formed from a tensegrity triangle with sticky ends. (B) Development of a DNA nanotube of undefined length using ssDNA tile lattice arrangement. (C) DNA hydrogel based on the pH-sensitive properties of i-motif. Acidification leads to assemblage of the Y-shaped scaffolds into a gel

development of biomimetic nanotubes devised from various biological macromolecules. DNA has shown multiple advantages in this respect due to its high programmability and well-defined knowledge of its structure. DNA nanotubes can be constructed from multiple building blocks such as single-stranded tiles, multicrossover tiles, DNA origami, and multi-rungs. ssDNA tiles are generally used to synthesize nanotubes of undefined length (Fig. 6.8B). Multi-crossover tiles can be used to manufacture DNA nanotubes of both defined and undefined length. These tiles are generated by an assembly of multiple four-arm junctions, DX tiles, and TX (triple crossover) tiles. DNA origami nanotubes are designed using two basic strategies, namely, direct self-assembly or by wrapping of rectangular origami. Finally, multi-rung stacking was proposed as a novel way to design DNA nanotubes. These nanotubes have a triangular or square cross section and are assembled using rungs made of cyclic DNA. The rungs are the basic building blocks which are fused via linkers to form the nanotubes (Liu et al. 2019a). DNA nanotubes serve as scaffolds for the precise assembly of several molecular compounds and can be

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used for diverse applications ranging from drug delivery to biosensing (Schaffter et al. 2020).

6.2.6

DNA Hydrogel

DNA hydrogels are synthesized by two methods which involve either direct chemical linkage of DNA or physical entanglement between multiple DNA chains. Chemical linkages involve the formation of covalent bonds between different chains, thus endowing the structure with superior stability and mechanical strength. In physical entanglement, the hydrogels are stabilized only by weak interactions such as hydrogen bonding, electrostatic interactions, and metal–ligand coordination. Both these kinds of hydrogels can be further categorized as pure and hybrid hydrogels. Hybrid hydrogels contain additional materials in the structure such as functional nucleic acids (FNAs) and peptides. These compounds are fused to the nucleic acid chains. The synthesis of hybrid hydrogels is a laborious process requiring multiple steps and longer times. To overcome these difficulties, pure hydrogels constructed entirely from DNA molecules were designed. The structure of pure DNA hydrogels often depends heavily on non-Watson–Crick interactions along with normal Watson–Crick base pairs. Additionally, enzymatic ligation and enzymatic polymerization are also routinely used to assemble these structures. Recent developments have led to the design of smart hydrogels made of DNA which can be regulated by specific stimuli. Both the assembly and stiffness of the hydrogel structure can be controlled by specific triggers which target a specific module of the hydrogel. For example, i-motif structures are popular choices for the development of hydrogels that are sensitive to pH. Since i-motif structures are only stable at an acidic pH, the hydrogels assembled from these structures form a gel only upon acidification of their environment (Fig. 6.8C). G-quadruplex/hemin-based DNAzymes are also used as functional units of DNA hydrogels for the colorimetric detection of target compounds (Khajouei et al. 2020).

6.3

Dynamic Self-Assembly Systems

Self-assembly is a nature of life. All living organisms assemble into the organized form necessary for life through the organization of scattered components. This process consumes energy and is dissipative. As long as life sustains, the requirement of energy never subsides and this energy works to always maintain a non-equilibrium state within living beings. Since the reaching of equilibrium would end the necessity for further change, living organisms must always avoid this state. So the process of life requires continuous active self-assembly. Artificial self-assembly systems generally are not active. They also begin from a scattered array of components, but the final state has a lower energy and thus is thermodynamically favoured. This kind of self-assembly does not require any energy input. To understand living systems it is necessary to create active self-assembly systems.

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DNA nanotechnology provides an amazing platform for the design of such active self-assembly systems. The particular advantage of DNA in this field is due to its extreme polymorphic nature. DNA can be directed to form a range of structural motifs simply by altering its sequence context. Dynamic DNA structures which are active in nature have also been developed which mimic some features of living beings. Dynamic DNA-based self-assembling systems can be categorized into three types: 1. A passive assembly–disassembly system which has two equilibrium states and alternates between them 2. Trigger-induced autonomous assembly systems which self-assemble upon stimulation by a specific trigger 3. Active (energy requiring) assembly systems involving a series of transformations

6.3.1

Passive Assembly–Disassembly System

As mentioned above the passive assembly–disassembly systems are characterized by two alternating equilibrium states. This is a very simple system which acts as a component in the construction of more complicated structures. The initial state of such a system is considered to be an equilibrium state. This equilibrium is disturbed by environmental changes or due to a stimulus. Once disturbed the system changes and achieves a new equilibrium state. When the environmental change is reversed or the stimulus is withdrawn, the system again reverses to the initial equilibrium state. DNA origami-based passive assembly–disassembly devices are often controlled by temperature. The system initially exists in a pre-assembled equilibrium state achieved by a process of thermal annealing. When this system is disturbed by increasing the temperature of the environment, the origami structures denature into its components, and this forms the second equilibrium state. Lowering of the temperature to previous condition recreates the initial structure. Other more complex assembly–disassembly systems may be controlled by two or more factors. These factors may include the presence or absence of cations and pH of the system.

6.3.2

Trigger-Induced Autonomous Assembly Systems

Unlike passive assembly–disassembly systems which have two equilibrium states (assembled and disassembled), trigger-induced systems have a single equilibrium state. In this case, the initial state is a non-equilibrium state which proceeds to an equilibrium when a trigger is present. An example of trigger-induced autonomous assembly system is the hybridization chain reaction (HCR). An HCR system consists of two hairpins, H1 and H2, both containing single-stranded toehold regions. The hairpins are designed such that each hairpin loop has the sequence complementary for the toehold of other hairpins. When a trigger DNA containing sequence complementary to one of the toehold and consecutive stem regions is added, one of the

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hairpins is unfolded. This unfolded hairpin binds to the toehold of the other hairpin which is thereby unfolded and a chain reaction begins. Multiple hairpin molecules are then unfolded and bind each other in a sequential fashion leading to the formation of a polymeric unit. A second example of this type of dynamic system is given by the catalytic DNA system. Here the trigger DNA is known as a catalyst as it is recycled once the equilibrium state is formed. Two forms of this system are summarized: 1. A substrate S is formed of three DNA sequences. Sequence 1 is the template strand, and the two other sequences are partially complementary to the template. All three sequences have toehold regions. The catalyst strand also binds to a part of the template and displaces one of the partially complementary strands such that a hidden toehold region in the template is exposed. A fuel DNA is then added that binds to the exposed toehold and displaces both the remaining partially complementary strand (output) and the catalyst. The duplex of fuel and template is the final remaining state referred to as waste. 2. The other form of this system is the chain hybridization system (CHA), which is somewhat similar to HCR. CHA also begins with two hairpins containing toeholds and stem domains with complementarity to inverse hairpins. The catalyst DNA binds and unfolds one of the hairpins exposing the regions complementary to the other hairpin. This leads to binding of one hairpin with the other. Interestingly, the complementary regions of the hairpin overlap the catalyst binding region, thus causing its release. The difference between HCR and CHA is in the binding patterns of the hairpins. In CHA, the two hairpins bind in such a manner that disallows the binding of other hairpins to this complex. The final type of trigger-induced self-assembly is exemplified by seed-guided self-assembly. This type of self-assembly allows spatiotemporal regulation of the self-assembly process. Here, the seed is a DNA origami which initiates the formation of self-assembled structures such as DNA tiles. In this context, it is important to note that self-assembly would occur even in the absence of the seed but the process would require significantly more energy thereby making it slower. The seed lowers the energy barrier so that the equilibrium is reached faster. Once the process begins, continuous growth occurs under isothermal conditions. Termination of the process can be achieved by using caps of varying rigidities that hinder the growth interfaces.

6.3.3

Active Assembly Systems

The systems discussed above always change towards an equilibrium state. In the equilibrium the system is stable unless the environment affecting the system is altered. In active assembly systems, devices work without ever reaching equilibrium. This is similar to the nature of life and thus extremely important to decipher life-like processes. Scientists have just begun to generate systems with such active properties, and some of the successful devices are discussed below.

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Information Transmitter Information relay is an important part of maintenance of cellular pathways to regulate important cellular processes. Transmission of information in a controlled manner in nanodevices has thus captivated the attention of scientists all over the world. Recently, a basic system has been developed that allows transmission of information in a DNA-based diamond-shaped structure. In this structure, four adjacent elements share a common edge which has a scissor-like opening function. A trigger DNA which serves as the information or signal leads to opening of this scissor. This structural change translates to the four associated elements leading to change in their structure as well. This further leads to change in the consecutive segments, which ultimately leads to a complete change in the structure. It is also possible to control this information relay in a spatiotemporal manner by controlling the exact location and time of adding the trigger DNA. By altering the internal elements, it is also possible to stop the transduction at specific points. This system undergoes a continuous oscillation of energy as the change of structures to different states requires energy. Each unit structure that allows information transduction from one side to other is the basic repeating unit of this structure, and the transition of this structure leads to the formation of a high-energy state. So, repeated formation of the high-energy states leads to oscillation of the graph in the energy landscape. Biochemical Oscillator-Regulated Dynamic Assembly System One of the most critical elements maintaining cellular homeostasis is spatiotemporal regulation of cellular events. For example, in a dividing cell cytokinesis must begin at a specific time and cleavage of membrane must occur at a specific location. Any deregulation in this control will damage the newly formed cells. Similarly the polymerization and depolymerization of microtubules in a specific orientation are central to their biological functions. Such spatiotemporal regulation is a product of a biochemical oscillator formed with cellular factors. Such a system can also be replicated in an artificial assembly. An example of a chemical oscillator is given by the BZ system. The BZ system exemplifies a chemical reaction which works out of equilibrium. The reaction itself has a series of steps and shows excitability. When an input is introduced to this system, the result reaches a maximum value and then drops to initial state. So, an oscillating system is formed, but it cannot be combined with other systems due to its extreme susceptibility (Dong et al. 2020). Artificial Metabolic System To develop this system, scientists have developed a DNA hydrogel which is synthesized and assembled into a gel by a microfluidic device. This gel forms defined structures by using energy. The catabolic process occurs simultaneously by a DNA-hydrolysing enzyme, and these processes can be controlled spatiotemporally by a separate inlet. This system can show locomotive and racing functions. The anabolic and catabolic mechanisms in this device mimic a metabolic system and are referred to as DASH (DNA-based assembly and synthesis of hierarchical materials) (Dong et al. 2020; Hamada et al. 2019).

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Examples of Dynamic DNA Nanorobots

DNA nanorobots have gained wide appreciation from the scientific community due to their compatibility with the biological system. Unlike conventional robotics, these machines can act specifically on target tissues in biological systems. Additionally, they can penetrate cells through specific interactions and thereby function in the cellular microenvironment with relative ease. A DNA nanomachine is characterized by four essential features: • • • •

The ability to perform a mechanical function such as translocation or rotation The necessity of a fuel that triggers the mechanical operation Utilization of energy leading to the formation of waste products Presence of anti-fuel to allow the continuous operation of the device (Wang et al. 2014)

So, these devices demonstrate the active assembly systems described above. Several DNA nanorobots have been designed in the past decade including DNA nanowalkers, DNA switches, DNA tweezers, and many others. Each of these machines is sensitive to different stimuli such as pH, presence of ions, or specific target proteins. These nanorobots are regularly used for cellular imaging, drug delivery, biosensing, and modulation of cellular pathways and in biocomputing. While the applications of DNA nanotechnology will be described in detail in later sections, here we will give a brief glimpse into the use of DNA nanorobots in various fields. Various DNA machines have been developed that allow cellular imaging by targeting various cellular features. Exemplary work in this field was performed by Yamuna Krishnan’s group. They used the pH-sensitive nature of the i-motif structure to design a probe for the pH mapping of biological cells. The C-rich unit in their device was functionalized with fluorescent dyes that could undergo FRET when their proximity was reduced. When the nanodevice encountered an acidic pH, the i-motif structure was formed which induced FRET. This device was named the I-switch and was later utilized for pH mapping of Caenorhabditis elegans. Further by using two nanorobots within the same cell which moved through the cell by different trafficking pathways, the authors were also able to map the pH of various cellular entry points within a single cell. This design termed the 2-IM was later employed to map both pH and Cl- ions by using Cl--sensitive dyes along with the pH sensing i-motifs. The 2-IM machine was also employed to image human fibroblasts from skin biopsies of normal and diseased individuals having the Niemann-Pick disease. Highly distinct lysosome subprofiles were generated by these machines allowing better diagnosis (Leung et al. 2019; Nummelin et al. 2020). DNA nanorobots bearing aptamers targeting specific proteins will target specific cells and may be used for therapeutic purposes. A DNA nanorobot tagged with an aptamer targeting nucleolin was developed for this purpose. Nucleolin is regularly overexpressed in cancer cells and offers a good target for cancer cell detection. The nanorobot used in this case contained thrombin within a DNA

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origami-based enclosure. For this purpose, the thrombin molecules were fused to poly-T oligonucleotides and a rectangular DNA origami sheet with poly-A extensions was generated. The thrombin moieties were thus attached to the origami sheet by Watson–Crick base pairs, and the rectangular sheet was then fastened by the fastener and targeting strands to form a nanotube. The fastener strands used had the aptamers for binding nucleolin. In the presence of nucleolin, the aptamers interact with it leading to uncoiling of the double helices fastening the rectangular sheet, thereby freeing the thrombin molecules. These then cause thrombosis of the carcinoma-associated blood vessels leading to necrosis of tumour cells. Nucleolin therefore serves both as a target and as a stimulus leading to reconfiguration of the nanorobot from the closed (thrombin shielded) to the open state (thrombin exposed) (Li et al. 2018). DNA origami has also been instrumental in the development of various drug delivery devices. For example, Li et al. have developed a DNA origami structure containing just 30 staple strands in addition to a 960 base scaffold strand. This ultrasmall device costs less than 100 dollars and is able to penetrate cancer cells when loaded with doxorubicin (Li et al. 2020). The use of DNA nanorobots in diverse fields can only be briefly discussed here. The functioning of some DNA machines is discussed in the following sections. Nanowalkers: The exceptional programmability of DNA has led to the establishment of nanowalkers which are the advanced DNA nanomachines that move gradually along tracks. Nanowalkers present in the living body such as myosin, kinesin, and dynein move gradually in a linear manner across the polar tracks. DNA nanowalkers are the artificial molecular machines that are designed for their unique programmability, predictability, and controllable nature (Chao et al. 2019). DNA nanowalkers can control their movements along the DNA footpaths in a progressive manner which can be one-dimensional (1-D), DNA origami (2-D), or particles (3-D) (Zhang et al. 2020). Each walking step is followed as payload release through hydrolysis or displacement of the substrate strand and also characterized at the micro- or nanometer scale. Endonuclease, exonuclease, and DNAzyme are commonly used to hydrolyse the substrate strands (Yang et al. 2016). There is a nanowalker powered by a duplex-specific nuclease (DSN) that can move independently and gradually on a spherical three-dimensional track created by hybridizing a 13 nm diameter gold nanoparticle (AuNP) with densely mismatched DNA duplexes (Chen et al. 2021). It has no affinity for double-stranded RNA (dsRNA) or singlestranded DNA (ssDNA) or RNA (ssRNA). DSN can only electively cleave the exactly matched dsDNAs or DNA–RNA hybrids that have at least 10 or 15 base pairs that further help to differentiate the partially or perfectly matched duplexes (Kim et al. 2017; Kuang et al. 2019). DSN cleaves the perfectly matched DNA– RNA hybrid while also releasing the walking strand. Then the walking strand moves independently through consuming mismatched DNA duplexes (Liu et al. 2019b). DNA walkers that traverse three-dimensional tracks have a greater capacity for simulating complex biological systems accomplishing effective signal amplification. This benefit has fuelled interest in DNA walkers with 3D tracks through the surface of different nano- and micromaterials such as carbon nanotubes (Cha et al. 2014), microparticles, and gold nanoparticles (Qu et al. 2017; Li et al. 2017).

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Nanotechnology aimed to invent rationally designed nanowalkers that act like biomotors that can transport the cargos independently (Sherman and Seeman 2004). Through recreating this system it is also possible to efficiently utilize the chemical energy at a single molecular level. These nanowalkers are primarily burnbridge motors that traversed the track in a domino-like cascade (Shin and Pierce 2004). One of the major hindrance remains in harvesting the chemical energy in a single molecule level (Toyabe et al. 2011). Indeed using nanowalkers can satisfy the criteria by using several chemo-mechanical gating mechanisms that are important for optimal fuel use and continuous motion. One of the examples of nanowalker is catalytic hairpin assembly that helps in walking on DNA coated microparticle surfaces that enable the detection of amplifiable microRNA (Jung et al. 2016). Imaging of intracellular base excision repair (BER) activity is also possible using DNA bipedal nanowalkers by monitoring human apurinic/apyrimidinic endonuclease 1 (APE1) activity. APE1 plays an essential role in base excision repair (BER) pathway through incision of DNA towards the abasic site and also helps in maintaining genomic integrity and fidelity (Vascotto et al. 2009; Tell et al. 2009). This bipedal nanowalker platform can be used for the advancement of signal amplification for high contrast imaging as well as for the investigation of low abundance biomarkers within living cell (Loh et al. 2014). DNA switches: Dynamic DNA nanotechnology includes various devices such as DNA circuits, motors, and switches. Switchable DNA nanostructures are mostly triggered by the local chemical changes such as pH or concentration and external actuation driven by light, electric, or magnetic fields. Nanoswitch: In the emerging area of synthetic nanotechnology, DNA is recognized as the ideal material for the nanoscale construction as the base pairing rule is easy to predict, also DNA hybridization is configurable. DNA synthesis is also easy and it can be chemically modified. The method of creating the custom DNA nanostructures belongs to DNA origami where a single-stranded DNA scaffold strand can be folded into any shape as per requirement (Seeman 1982). These strands are further crosslinked by the staple strands. DNA origami nanostructures can be made functional by attaching single-stranded DNA “handles” to the ends of staple strands on the structure’s surface as each staple sequence is distinct; handle locations can be addressed in a distinct manner (Rothemund 2006). There are different guest molecules like fluorophores, proteins, or metallic nanoparticles that are attached to the complementary anti-handle sequences. So the changes in the environment may act as a switch to change the nanostructures from one to another form (Douglas et al. 2009). This switch could be used to initiate nanostructures to carry out specific tasks such as helping to bring proteins together to quantify their interaction, shifting the chirality of a plasmonic device (Kuzyk et al. 2014), or induction of opening of capsule to deliver the molecular cargo (Wu and Pauly 2022; Douglas et al. 2012). Other factors such as change in pH (Surana et al. 2011), ionic concentration (Burns et al. 2018), or addition of DNA nucleotides or proteins may act as trigger for the molecular switch. Currently, researchers have made strides towards creating switchable DNA nanostructures that respond to

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Fig. 6.9 Strand displacement-based switchable transitions in a DNA tweezers structure showing building and use of the molecular tweezers

external cues such as light and electric or magnetic fields (Lauback et al. 2018; Kroener et al. 2017). Nanotweezer: It is a structure that consists of two arms connected via a DNA linker which can transit between open and close states in response to external cues. This construction leads to the formation of a nanomechanical switch. This machine can use DNA not only as a structural material but also as a fuel as fuel (Fu) and antifuel (aFu) mechanism (Yurke et al. 2000; Wang et al. 2014). As described in the image the tweezer contains the A and B construct (Fig. 6.9). The A construct contains two arms named as a and b connected by the complementary c strand. The arms contain tethers T and T′ that are single stranded and help in the formation of open construct. The closure of the tweezers proceeds to yield the closed structure, construct B, in the presence of the fuel strand F, which is complementary to the tether domains T and T′ of a and b. The merging fuel strand (F) is displaced by the anti-fuel strand, aFu (F′), leading to the formation of fuel/anti-fuel duplex that is energetically stabilized resulting in the opening of the tweezers. The cyclic mechanical switching of the tweezers structure between the open and closed states was followed by the fluorescence intensities of the fluorophore that are controlled by the distance separating the fluorophore/quencher pair by labelling the 5′ and 3′ends of the bridging strand (c) with a fluorophore/quencher (F/Q) pair. In the open state of the molecular device, the spatial separation of the fluorophore/quencher pair results in high fluorescence; meanwhile in the closed state the contact between fluorophore and quencher results in enhanced quenching of the fluorophore (Wang et al. 2010).

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Effects of microenvironment on switching devices: Ionic concentration: Ionic concentration change may act as a switch for nanomechanical devices. High salt concentration may act as a switch in conversion of right-handed helix (B-DNA) to left-handed (Z-DNA) structure (Jones et al. 2015). G-quadruplex structures containing four G quartets which are bounded by the cyclic Hoogsteen hydrogen bond are stabilized by the monovalent cation such as Na+, K+, or Li+ (At 2010). DNA origami nanostructures are also sensitive towards the divalent ion concentration and can change their structure according to the changing ion concentration. Based on shape complementarity, blunt end stacking of DNA helices could be used for hierarchical arrangement of DNA origami nanostructures into larger crystals (Yang et al. 2012). Introducing two guanine strands into a DNA nanostructure can form an inter-strand G-quadruplex structure in the presence of K+ and break down in the absence of K+, so it can act as nanoswitch (Kuzuya et al. 2011). pH sensor: pH sensor devices such as triplex or i-motif structure can act as switchable DNA nanostructures as their stability relies on acidic pH (Gerweck and Seetharaman 1996). Nanoswitch as pH sensor has great in vivo applications in cancer tissue as acidic conditions can be seen in endosomal pathway (Idili et al. 2014). These DNA structures form duplex with the complementary strand at neutral or alkaline pH. Triplex structure (C+-GC) forms during pH cycling between 5.0 and 8.0, but at pH 8 three strands individually form three duplexes, one arm of the crossDNA binds to a ssDNA on the other arm, forming a DNA triplex that locks the origami structure, and one of the single-stranded region binds to one of the duplex (Chen et al. 2004). These triplexes possess CGC pairings that are only stable at acidic pH (pKa 6.5) because protonation of the ssDNA cytosine at N3 position pH is required. Triple helix molecular switch (THMS) belongs to the analytical platforms that are widely used as DNA-based sensors for different targets (Jones et al. 2015) (Fig. 6.10). To avoid G4 formation, the i-motif-based nanodevices use C-rich sequences as motor DNA and a complementary sequence with very few mismatches. The i-motif structures are composed of intercalated hemiprotonated C.C+ base pairs which demands protonation at N3 position of cytosine and switching is triggered at acidic pH (Benabou et al. 2014). In this way, several DNA nanostructures can be obtained through pH switching. For example, DNA origami pliers were created by Kuzuya et al., who created a 170 nm lever structure embellished with cytosine-rich ssDNAs leading to the formation of i-motifs at acidic pH (Kuzuya et al. 2014). It was discovered that attaching a C-rich motor to a gold-coated array of micromechanical cantilevers where the nanoscale surface forces change in conformation of macroscopic effect resulted in bending of cantilever caused by the pH-driven change (Shu et al. 2005). Jiang’s lab created a nanocontainer for controlled drug delivery and release through linking C-rich DNA via a long linker to gold surface (Mao et al. 2007). An i-motif-based DNA nanomachine combined with fluorescence resonance energy transfer (FRET) measurements can be used to map temporal and spatial pH changes as a result of endosomal maturation in various Caenorhabditis elegans strains discovered by Surana et al. It was the first time a DNA nanomachine was

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Fig. 6.10 pH-driven transition of DNA nanostructures. (a) Under basic pH, the device’s three DNA strands form three duplexes. At an acidic pH, a single-stranded region is intended to bind to one of the duplexes, thus leading to the formation of triplex structure. (b) At high pH, C-rich DNA is bonded to a complementary strand and immobilized on a cantilever. By increasing DNA strand repulsion and forming an i-motif, lowering pH causes compressive surface tension, which is relieved by bending the cantilever

successfully employed and activated in an organism (Surana et al. 2011). The in vivo application of triplex structure as biosensor was used to track pH changes in living cells along with apoptosis in association with graphene oxide (Li et al. 2013). Effects of external stimuli on switching devices: Photoactivation: Ultraviolet radiation or visible light can act as stimulus for photoswitching such as the photoswitching of azobenzene linked to the DNA backbone which is the most prevalent approach. Photoregulation is possible through ultraviolet radiation according to Asanuma et al. When exposed to UV radiation (300–400 nm), azobenzene converts to a cis-form, which prevents DNA hybridization. When exposed to visible light (>400 nm), the azobenzene acquires a trans-form, allowing the production of a stable DNA duplex (Asanuma et al. 2007). Yang and colleagues created photoresponsive DNA origami hexagons by attaching them to azobenzene-modified oligonucleotides. Through tampering with the number and arrangement of Azo-ODNs on the origami, various origami hexagons could be linked as units. Azo-ODNs are photoactivated together to form larger two-dimensional structures in a variety of shapes (Yang et al. 2012). Electrical activation: As the phosphate backbone of DNA contains a net negative charge which causes the formation of DNA origami nanostructures, the electric field can be used as stimulus. Electrical switching is possible in the case of DNA origami nanolever on the gold surface as described by Kroener and colleagues (Kroener et al. 2017).

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Modifications of DNA in Nanostructures

To endow inert nucleic acid nanostructures with functional properties, modification of the DNA backbone often becomes necessary. The conjugation of DNA with other materials was proposed long back by Seeman in his earliest works on DNA nanotechnology. However, efforts to functionalize DNA molecules have only gained momentum in the last decade as scientists have realized their potential applications in advanced technologies. This has led to the commercial production of a wide range of modified oligonucleotides that are commonly used to control the chemical reactivities of DNA. These compounds regulate the formation of covalent bonds, oligomerizations and polymerizations, cross-linking, and metallization of the nanostructure. For the synthesis of a nanostructure containing functional moieties, the individual DNA strands first have to be modified. Pre-modified nucleosides can directly be incorporated during DNA synthesis or modifications can be attached post-synthesis using specific moieties. The modified oligonucleotide strand thus created can be incorporated into the DNA nanostructure by two methods, as is discussed below in the case of a DNA origami: 1. Direct integration: A DNA origami structure includes multiple short strands (staple strands) annealed to a long strand (scaffold strand). In the direct integration method, the modified strand can be directly incorporated as one of the staple strands into the structure. This allows better spatial control as the modified strand is completely embedded in the structure and is also easier since a single hybridization step is required. The limitations of this method are that the higher temperature required for DNA annealing may cause degradation of some agents that are regularly attached to DNA. For example, proteins and nanoparticles are often degraded during DNA denaturation before annealing. Also if several modifications are to be incorporated, separate modified strands are required for each conjugate. 2. Added to the nanostructure post-folding: As is suggested by the title, in this method the modified oligonucleotide is added to the origami structure postfolding via extensions on the staple strand. This method allows the attachment of multiple modified strands at various positions along the origami structure. This attachment occurs at room temperature preventing any degradation, but this type of integration has reduced spatial control as the modified oligonucleotide is extended from the origami structure. Additionally, the integration at room temperature is less efficient than the annealing process. Both of these methods are used depending on the nature of the conjugate and the required design of the nanostructure. We will now discuss some of the common conjugates used in DNA nanotechnology. Fluorescent Dyes and Quenchers Chromophores, in particular fluorophores, are the most frequent choice as conjugates in DNA nanostructures. These compounds are used for a variety of applications, and many chromophores are thus commercially

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available for these purposes. These compounds can be added to the nanostructures directly during synthesis or post-synthetically via appropriate handles. The postsynthetic addition mechanism is often favoured to avoid degradation of the fluorophores during the synthesizing process. In addition to fluorophores, quencher compounds are also widely available. These quencher compounds are used in association with a partner fluorophore whose fluorescence they can effectively quench by a transfer of energy from the fluorophore. The quencher then loses this energy by a non-radiative process. Additionally, multiple fluorescent dyes can be incorporated into the DNA nanostructure for FRET (Förster resonance energy transfer), which has sensitive applications in monitoring dynamicity and calculation of distance. FRET requires the use of two compatible dyes, namely, the donor and acceptor. The donor fluorophore is excited at a lower wavelength, and this energy is then transferred to a compatible acceptor. The excited acceptor then fluoresces at a higher wavelength. Here, it is important to note that the acceptor dye normally absorbs light in higher wavelengths compared to the donor, but during FRET it is excited by a non-radiative transfer of energy (Madsen and Gothelf 2019). The recent development of FRET and its usefulness in DNA nanotechnology are understood by the following example. Filius et al. have recently designed a nanostructure which allowed the measurement of multiple distances between distinct FRET pairs on a single object. Their device makes use of DNA exchange to obtain single-molecule FRET for multiple FRET pairs in a single nanodevice. Single-molecule FRET is an extremely sensitive technique that allows the determination of biomolecular conformations at the molecular level. While many other sensitive techniques like cryoelectron microscopy and nuclear magnetic resonance (NMR) also provide data at the molecular level, these techniques often ignore minute amounts of the rare conformations. Single-molecule FRET can give information about even the rarest conformational variant with subnanometer resolution. Although highly sensitive, the use of this technique is limited as it is difficult to calculate the distance between more than one or two FRET pairs simultaneously. Nanostructures have been developed that allow the measurement of distance between various FRET pairs to generate libraries that will allow the determination of biomolecular conformations using this technique. Various techniques have been applied to the generation of such libraries. These include switchable FRET which uses a single donor and multiple photoswitchable acceptor fluorophores such that only a single acceptor is active at any instant and point accumulation in nanoscale topography (DNA-PAINT) which allows the association between donor strand and acceptor strand to occur for a limited time only. The nanodevice designed by Filius et al. used the process of DNA exchange to allow the formation of a unique FRET pair transiently within a single molecule. This allows the calculation of the distance between the FRET pair formed and DNA exchange and then causes formation of a separate FRET pair. The higher spatial resolution in this technique is obtained by the DNA exchange using multiple DNA imager strands which could hybridize to the acceptor strand for several minutes before the system is washed. Then a second imager strand is introduced, and in this way, the distance between the multiple FRET pairs can be measured. This system was successfully integrated to DNA nanostructures having a

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triangular shape, which could then efficiently determine the distance between specific points placed on the vertices of the triangle. One vertex of the triangle was attached with a fixed point of interest (POI A) containing a donor strand; another vertex contained a different point of interest (POI B) with a separate donor. The acceptor strand is placed attached to a unique site which avoids photobleaching. It was observed that the FRET efficiencies for the different FRET pairs in this structure were altered significantly upon changing the linker length by a single base pair. Further, this nanostructure was also applied to the measurement of distances between three FRET pairs demonstrating the potential of the system (Filius et al. 2021) (Fig. 6.11a). Azobenzenes and Other Photoreactive Compounds In addition to serving as switch on or switch off probes, chromophores are also employed to alter the conformation of DNA nanostructures. This is made possible by the use of protecting groups which are released upon exposure to light. Alteration of structure by phototriggers is kinetically up to 60 times faster than strand displacement reactions. Compounds like azobenzene serve as photo-sensitive probes which can be directly incorporated into DNA. When azobenzene is exposed to UV light, it switches from the trans to cis conformation. A DNA duplex can be denatured by incorporating about 6 azobenzenes in the DNA structure. The azobenzenes are triggered by light to switch from trans to cis state. Since the cis conformation is non-planar, the duplex is denatured (Madsen and Gothelf 2019). This property of azobenzene has made it extremely popular in the synthesis of DNA-based nanostructures. Another compound with properties similar to azobenzene is arylazopyrazoles (AAP). Both AAP and azobenzene can be fused to DNA through a d-threoninol linkage. Recently, these compounds have been used simultaneously in the development of DNA origami structures with two photoswitches. In an exemplary study, AAP was individually used to develop an X-shaped and hexagon-shaped (H-shaped) origami. The AAP molecules undergo reversible transition from trans to cis state upon irradiation with 365 nm radiation, and the transition is reversed by light at 520 nm. For azobenzene, the reversing wavelength is 465 nm. Initially, an X-origami and H-origami were designed with the AAP linkages. A single edge of the X-origami has four AAP-modified oligonucleotides, and one of the edges of the H-shaped origami had complementary strands also modified with AAP. When irradiated with 365 nm light, these monomeric origami structures are unable to dimerize due to the cis-form of AAP, but upon exposure to 520 nm light, duplexes are formed between the complementary strands of X and H monomer leading to the formation of the XH dimer. This system could be assembled and disassembled reversibly by alternating the wavelength of light (Fig. 6.11b). The X-monomer was also used to form a tetramer by using AAP-DNA strands. In this case, the original and complementary AAP strands are placed on perpendicular edges of the X-monomer, and upon exposure to 520 nm light, four monomers hybridize to form the tetrameric structure (Fig. 6.8b). By changing the positions of the complementary strands to the opposite edge, linear arrays of the X-origamis were also generated (Fig. 6.8b). Finally, a Y-shaped DNA origami structure tagged with azobenzene containing strands was

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Fig. 6.11 Examples of fluorophores and photoreactive compounds tagged to DNA as functional groups. (a) Demonstration of the procedure used for measurement of distances between three FRET

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associated with the previously described X- and H-monomers to design a trimer containing three different steps. For this purpose, the H-origami was designed to have unique edges complementary to both the X-origami (having AAP-DNA strands) and the Y-origami (having Azo-DNA strands). The conjugates in these three monomers were initially all in the trans state so a significant amount of the XHY trimer was formed upon mixing followed by incubation of the monomers. Upon irradiation of this mixture with 365 nm light, the monomers were all separated. Further irradiation with 520 nm light allowed the formation of the XH dimer, but only a minute amount of the trimer is formed. Conversely upon exposure to 450 nm light both the dimers and the trimer were formed in significant quantities by the formation of duplex structures (Fig. 6.11c). This work suggests how lightsusceptible conjugates can be used to generate multiple forms of DNA nanostructures (Mishra et al. 2021). Hydrophobic Modifications Hydrophobic groups are attached to DNA nanostructures to serve as probes for interaction with the phospholipid membranes of living cells. These groups allow the nanostructure to cross the membrane barrier to control cellular functions. A wide range of hydrophobic moieties such as cholesterol, stearyl, tocopherol, diacyl, and multichain lipids are fused to DNA sequences. Most of these compounds are inserted during DNA synthesis as post-synthetic modification with these compounds is often complicated by the dual hydrophilic and hydrophobic character of DNA structures. A variety of compounds have been synthesized by hydrophobic modifications of DNA such as lipid vesicle to monitor and regulate cellular behaviour. Additionally, these compounds show a great potential as drug delivery devices (Madsen and Gothelf 2019). The discussion of the great range of nanodevices developed using hydrophobic moieties is beyond the scope of this book, but here we will outline an example of how hydrophobic moieties are used in DNA nanostructures. Molly M. Stevens’s group has recently succeeded in synthesizing a DNA nanoflower which is able to perform ratiometric aptasensing operations within tumour cells. This device makes use of DNA aptamers for specific tumour targeting along with sensing of target analyte. Aptameric sequences are often compared to antibodies in their action as they can bind target compounds with high affinity and selectivity. A DNA aptamer tagged with cholesterol moieties has been used for efficient cellular uptake. The use of cholesterol along with a cell-membrane

 ⁄ Fig. 6.11 (continued) pairs in a single molecule. The acceptor strand is placed attached to a unique site which avoids photobleaching. The distance between POI B and the acceptor can be altered by a modifiable linker and even single base pair changes could be efficiently detected. (b) Formation of XH dimer, X-tetramer, and linear X-arrays from X-shaped and/or hexagon-shaped DNA origami nanostructures functionalized with AAP strands. (c) Development of an XHY trimer by using AAP and azobenzene functionalized strands. The trimer once formed could be broken into monomers by 365 nm light irradiation. Further irradiation with 520 nm light allowed the formation of the XH dimer, but only a minute amount of the trimer is formed. Conversely upon exposure to 450 nm light both the dimer and the trimer were formed in significant quantities

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Fig. 6.12 Cholesterol-tagged DNA nanoflower which is able to perform ratiometric aptasensing. This device makes use of DNA aptamers for specific tumour targeting along with sensing of target analyte. A DNA aptamer tagged with cholesterol moieties has been used for efficient cellular uptake. The use of cholesterol along with a cell membrane targeting aptamer simultaneously acts to facilitate cellular uptake. This designer device makes use of rolling circle amplification (RCA) using a circular template DNA. Once within the cell, the target molecule displaces the acceptor-tagged DNA strand leading to a change in the fluorescence emission

targeting aptamer simultaneously acts to facilitate cellular uptake. This designer device makes use of rolling circle amplification (RCA) using a circular template DNA. This template is amplified using a DNA polymerase to produce a continuously repeated polymer of the complementary strand to the template. This complementary strand has 3 sequence domains repeated continuously. Domain 1 encodes the celltargeting aptamer; domain 2 has three sub-regions which are complementary to short oligonucleotides containing a donor and acceptor for FRET. The third sub-region of domain 2 is the target binding domain, and a part of the acceptor DNA binds this region. Finally, domain 3 acts as a complementary region for binding the cholesterol-modified DNA aptamer. The repeated complementary element thus generated leads to the loss of pyrophosphate ions which combine Mg2+ present in the buffer to form crystals. This crystallization process results in the packaging of the DNA amplicons resulting in the formation of DNA nanoflowers (DNF) after 6 h. During this time, the cholesterol-DNA aptamer is added to the mixture to develop CnDNF, and finally, the donor and acceptor strands are hybridized to form the functional CnDNF aptasensor. This aptasensor once incorporated into the cell can sense concentrations of the target analyte. The target binding aptamer of domain 2 binds the analyte which displaces the acceptor strand. So, FRET cannot occur and the emission wavelength from the crystal changes. This change is monitored by a ratiometric approach tracking the changes of FRET efficiencies from high to low (Kim et al. 2021) (Fig. 6.12). Biotinylation of DNA Biotinylation of DNA is commonly used to tag proteins to DNA through a non-covalent bond. Biotin (vitamin B7) interacts with the tetravalent avidin proteins through strong non-covalent bonds. The most commonly used avidin protein used for protein modification is streptavidin. Biotin can be added to DNA

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Fig. 6.13 (a) DNA nanogel containing multiple active enzymes. Biotinylated DNA fragments were synthesized by PCR of template DNA using varying percentages of biotinylated dCTP. When streptavidin-tagged enzymes were added to this mixture of bt-DNA, multiple cross-links developed due to the interaction between biotin and streptavidin. (b) The redox properties of the G-quadruplex/hemin complex were used to quantify target molecules. For this purpose, a tetrahedral nanostructure composed of four DNA strands was synthesized. Three of the sequences have a thiol group at the 5′-end which is used to link the tetrahedron to an Au-electrode. This nanostructure has two states, namely, relaxed and taut. In the relaxed state, there is no target and the aptamer is free. In the presence of target, the aptamer binds it leading to a decrease of the distance between the halves of the split G-quadruplex

during or post-synthesis at various locations (5′, internal, or 3′), and biotinylated DNA is also commercially produced (Madsen and Gothelf 2019). Recently, Mariconti et al. have developed a DNA nanogel containing multiple active enzymes by making use of the biotin–streptavidin interaction. The authors prepared biotinylated DNA fragments using PCR. For this purpose, the template DNA (without biotin) was amplified in the presence of varying percentages of biotinylated dCTP. The higher the percentage of bt-dCTP used, the more was the frequency of the biotin tag in the amplicon. When streptavidin was added to this mixture of bt-DNA, multiple cross-links developed due to the interaction between biotin and streptavidin. The size of the nanogel thus formed depends on the concentration of streptavidin added, and the authors tested various concentrations of streptavidin to calculate the amount of streptavidin required to generate the most compact forms of the nanogel. The streptavidin moieties were further used to introduce enzymes within the nanogel structure. The authors have successfully incorporated active alkaline phosphatase or horseradish peroxidase into the nanogel structure simply by conjugating the enzymes with streptavidin and mixing with bt-DNA followed by incubation on ice. Additionally, β-galactosidase, a very large tetrameric protein, was also incorporated into the nanogel in its active form by using the biotin–streptavidin linkage. Further, a multi-enzyme system was developed using streptavidin moieties tagged with different proteins. This system allowed coupling of different enzymes (Fig. 6.13a). For example, glucose when oxidized by glucose oxidase led to the

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formation of hydrogen peroxide which was oxidized by horseradish peroxidase to form an enzyme cascade (Mariconti et al. 2021). Redox-Active Modifications Functionalization of DNA nanostructures with electrochemical groups allows sensitive detection of signals through cyclic voltammetry. Commonly used compounds such as methylene blue, ferrocene, porphyrin, and pyrin chromophores have been added to DNA for this purpose. Addition can be made both internally and at the terminal positions. Metal-coordinating moieties have also been inserted into DNA as they are endowed with electron-transfer abilities (Madsen and Gothelf 2019). Recently, the redox properties of the G-quadruplex/ hemin complex have been used to quantify the amount of ATP. For this purpose a nanostructure composed of four DNA strands was synthesized. These strands form a tetrahedron, and three of the sequences have a thiol group at the 5′-end. These thiol groups appear at the same edge of the tetrahedron upon assembly and are used to link the tetrahedron to an Au-electrode. Of the six edges of the tetrahedron, five remain in the duplex state. The remaining edge has sequences embedded which form an aptamer for ATP detection. Additionally, it has sequences which form a split G-quadruplex. The tetrahedral DNA nanostructure thus formed has two states, namely, relaxed and taut. In the relaxed state, there is no ATP and the aptamer is free. In this state, the distance between the two halves of the G-quadruplex is large and the quadruplex is unable to form. In the presence of ATP, the aptamer binds it leading to a decrease of the distance between the halves of the split G-quadruplex. This forms the taut state of the nanostructure, and the addition of hemin in this state causes the formation of G-quadruplex/hemin DNAzyme, which can be measured using a differential pulse voltammogram (DPV) (Jing et al. 2020) (Fig. 6.13b).

6.5

Nucleic Acid Analogues

Natural nucleic acids although extensively useful in the design of versatile nanostructures may be difficult to use in a cellular context due to their nuclease sensitivity. This led to the development of various nucleic acid analogues which form double helices with ssDNA to form a much more stable structure with reduced susceptibility to nucleases. Additionally, the charge developed by these analogues is different from normal dsDNA allowing the introduction of electrostatic variations in the nanostructure. These may facilitate internalization of the nanostructure within the cell. In view of these advantageous properties, we will discuss in brief some forms of nucleic acid analogues and their use in nanotechnology (Madsen and Gothelf 2019). Peptide Nucleic Acids (PNAs) PNAs were first discovered by Nielsen et al. and are composed of a backbone constituted of repeated units of N-(2-aminoethyl) glycine. The purine and pyrimidine nucleobases in this structure are connected through a methyl carbonyl linker. The backbone of PNAs is uncharged, and thus these polymers can easily hybridize with DNA or RNA sequences having complementarity. PNAs can also be fused to functional groups so as to allow the design of

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nanostructures with specific functions (Pellestor and Paulasova 2004). The ability of PNAs to recognize and bind complementary sequences with high strength has led to their wide use in nanotechnology. PNAs have often been used as anti-sense oligonucleotides and for purposes of drug delivery. For example, Lee et al. have developed a microRNA-responsive photodynamic therapeutic platform for the specific killing of cancer cells. This device consists of a PNA sequence complementary to miR-21, which is often overexpressed in cancer cells. The PNA is tagged with a chlorin e6 (Ce6) moiety which acts as a photosensitizer and kills cancer cells by emitting radiation at the near-infrared region. In the nanostructure, the Ce6 fluorescence is initially inactivated by embedding it on a dextran-coated reduced graphene oxide nanocolloid (Dex-RGON) surface. The graphene oxide initially quenches Ce6 radiation, but when the PNA binds miR-21, the PNA/Ce6 conjugate is freed from Dex-RGON allowing fluorescence enhancement and tumour cell killing (Lee et al. 2016). Locked Nucleic Acids (LNAs) These are also known as bridged nucleic acids (BNAs) as there is a methylene bridge between the 2′-O and 4′-C atoms of the ribose molecules constituting the polymer. The formation of this bridge locks the structure into a rigid conformation; thus, these molecules are often also referred to as inaccessible RNA. The formation of this methylene bridge also imposes entropic constraints which makes the binding of complementary strands by LNA more favourable. LNA strands are resistant to nucleases and show good solubility in aqueous media (Lundin et al. 2013). A novel drug delivery system was generated using a nanoparticle assembly containing star-shaped glucose-core polycaprolactone-polyethylene glycol (Glu-PCL-PEG) block copolymer. Polycaprolactone is a polyester that is hydrolysed in physiological conditions through degradation of its ester bonds. This assembly was functionalized with the aptamer AS1411 which targets the protein nucleolin. Nucleolin is overexpressed in a large fraction of metastatic cells and thus serves as a good target. The nanoparticle assembly also contains a LNA which has a sequence complementary to miR-214. miR-214 shows unregulated expressions in various cancers and may augment both tumorigenic and tumour-suppressive pathways. Cisplatin is also embedded in this drug to sensitize cisplatin-resistant cancer cells. The drug is internalized through the function of glucose transporters (GluTs). Since cancer cells are often in a hypoxic environment, they show high levels of glycolysis. To obtain adequate energy from glycolysis, large amounts of glucose have to be transported to the cell. This causes the overexpression of GluTs in the cancer cell. Once internalized, the drug causes reduction of miR-214 activity and releases cisplatin causing death of cancer cells (Vandghanooni et al. 2020). Phosphorothioate-Modified DNA (psDNA) The phosphorothioate modification of nucleic acids was one of the earliest modifications explored to develop nuclease resistance properties. In psDNA, a single oxygen atom of the phosphorus is replaced with sulphur. This imposes a chirality on the phosphorus group which makes the DNA less susceptible to nuclease (Madsen and Gothelf 2019). In addition to creating

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probes for targeted gene silencing operations (Deleavey and Damha 2012), psDNA has also been utilized to stabilize fluorescent gold and silver nanoclusters. The psDNA backbone interacts more strongly with the metal nanoclusters, and it was observed that AgNCs template on psDNA shows altered properties compared to unmodified DNA (Weadick and Liu 2015). Other DNA and RNA modifications have also been developed through the years. Most nucleic acid analogues are used for their nuclease resistance. They are used most commonly as drug delivery agents and as anti-sense oligonucleotides.

6.6

Applications of DNA Nanotechnology

One of the most promising applications of structural DNA nanotechnology is drug delivery and therapeutics. In this aspect, artificial nucleic acid nanodevices that sense their surroundings could be used to deliver targeted drugs to tissues. Tissue Engineering The most common tissue transplants include bone grafts with 2.2 million cases being performed worldwide yearly. The technique cannot be accomplished successfully in several cases due to donor site morbidity and limited supply. These failures have led to the development of nano-engineered particles and 3D scaffolds that gained attention for enhancing the growth of new bone (Krishnan and Seeman 2019). Natural bone is made up of organic collagen fibrils and calcium phosphate crystals. To successfully regenerate on a macro- and nanoscale, this composition helps to mimic the bone structure (Madsen and Gothelf 2019; Zietz et al. 2013). The size of the nanoparticles ranges from 200 nm sized particles are phagocytosed and eliminated through spleen (Verdun et al. 1990; Fernández-Urrusuno et al. 1996; Rolland et al. 1989). The surface feature of bone is almost 100 nm in size. Natural bone surfaces frequently have features that are about 100 nm in size (Shi et al. 2010). The body would try to reject an artificial bone implant if the surface was left smooth. Due to this smooth surface, a fibrous tissue covering the surface of the implant is likely to form. The bone-implant contact can be further reduced, which may result in implant loosening and additional inflammation. Through putting nano-sized features on the surface of a hip or knee prosthesis, one could improve its performance by reducing the likelihood of rejection and excite the osteoblasts production. The osteoblasts are the cells that leads to the formation of the bone matrix discovered on the developing bones’ advancing surface (Salata 2004; Gutwein and Webster 2002). Titanium is regarded as a well-known bone-repair material that is mostly used in orthopaedics and dentistry as it possesses excellent fracture resistance, ductility, and optimal weight-to-strength ratio. Although it suffers from bioactivity, it does not promote effective cell adhesion and growth (Takizawa et al. 2018). The exact nanoscale mechanism that results in this useful combination of properties is still being debated. Ceramic nanoparticles and poly (methyl methacrylate) copolymer were used to create an artificial hybrid material which is about 15–18 nm (de la Isla et al. 2003; Roy et al. 2003).

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Cancer Therapy Advanced therapeutic procedures are urgently required for cancer as it is one of the leading causes of death in the globe. An important push to enhance medicine delivery in cancer has been made possible by the development of new nanomaterials and nanocarriers. Since structural and physical traits including size, shape, surface, and charge properties govern the biodistribution and other pharmacokinetic profiles including drug administration and safety of medication, rational nanoparticle design is essential. Contrarily using nanocarriers as a therapeutic agent may provide several advantages such as offering superior pharmacokinetic features, prolonged blood circulation time, distribution volume, cellular uptake, and half-life, which are crucial for greater therapeutic potential and achievement in clinical aspects (Aslan et al. 2013). For cancer detection, nanoparticles use the cancer biomarkers such as exosomes, circulating tumour cells, and nucleic acids or proteins associated with cancer (Jia et al. 2017). Nanoparticles have a high surface area to volume ratio in comparison to bulk materials which is a key benefit of using them for cancer diagnosis (Song et al. 2010). This characteristic allows for the dense coating of nanoparticle surfaces with antibodies, peptides, aptamers, small molecules, and other components. These moieties have the ability to bind and identify particular cancer molecules. Multivalent effects can be produced by exposing cancer cells to a variety of binding ligands, which can increase an assay’s specificity and sensitivity (Kumar et al. 2017). Several miRNAs, carbohydrates, and nucleic acids act as a biomarker for cancer (Ma et al. 2015). High selectivity, sensitivity, and the capacity to perform several target measurements simultaneously are all provided by nanotechnology. Nanoparticles and nanomaterials can be used to enhance biosensors to offer precise targeting (Sharifi et al. 2019). Three popular nanoparticle probes for the detection of cancer are quantum dots (QDs), gold nanoparticles (AuNPs), and polymer dots (PDs) (Harun et al. 2013; Wang and Jia 2018). Cancer biomarkers have been found using QD-based biosensors. The distinctive qualities of QDs include their extensive absorption with narrow, elevated Stokes shifts, high molar extinction coefficient, quantum yield, and exceptional resistance to deterioration (Medintz et al. 2005). A sandwich immunoassay for ZnO nanowire substrates which is based on ZnO QDs was developed because they offered a significant surface area and along with several binding sites used for detection purpose (Gu et al. 2011). The foundation of photodynamic cancer therapy is the cytotoxic atomic oxygen produced by lasers that kill cancer cells. The amount of a specific dye that is used to produce atomic oxygen is taken up by the cancer cells in greater amounts than it is by healthy tissue. As a result, mainly cancer cells are eliminated after being exposed to laser radiation. Sadly, the leftover dye molecules move to the skin and eyes, making the patient extremely sensitive to sunlight. Up to six weeks may pass before this effect fades (Yan and Kopelman 2003). The hydrophobic dye molecule was contained inside a porous nanoparticle to prevent this negative effect. The dye was contained by the Ormosil nanoparticle and did not diffuse to other body parts. The oxygen was able to diffuse out easily due to the pore size of roughly 1 nm, and its ability to generate oxygen was unaffected (Roy et al. 2003). There are different nanoparticles causing the enhancement of MRI contrast such as iron oxide-based

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nanoparticles (Perez et al. 2003; Yan et al. 2004), gadolinium-based nanoparticles (Oyewumi et al. 2004), and multiple-mode imaging contrast that connect biological targeting and optical detection with magnetic resonance (Levy et al. 2002; Bergey et al. 2002). Nanoparticle probes containing molecularly targeted recognition agents have the potential to reveal the presence, relative abundance, and distribution of cancer signatures and markers associated with the tumour microenvironment (Ma et al. 2018). Crosslinked iron oxide nanoparticles were used for MRI identification of camptothecin-induced apoptosis of Jurkat T cells in vitro because they were tethered to annexin-V, which identifies the presence of phosphatidylserine on apoptotic cells (Schellenberger et al. 2002). Angiogenesis is an important marker for the detection of cancer cell. MRI has been used successfully by several groups to image angiogenesis in animal models using different formulations of derivatized nanoparticles targeted by αvβ3-integrin (Winter et al. 2003a, b; Sipkins et al. 1998). The ability of MRI to detect signals from epitopes targeted by appropriate nanoparticles at extremely low picomolar concentrations offers hope for potential clinical applications (Morawski et al. 2004). The ability of nanotechnology to “multiplex”, or detect a wide range of molecular signals and biomarkers in real time, is what propels advances in early detection, diagnostics, prognostics, and therapeutic strategy selection. Multiplexing detection nanotechnologies include arrays of nanocantilevers, nanowires, and nanotubes (Yue et al. 2004; Chan et al. 2002). In the case of ex vivo biomarker detection, nanoparticles show great potential; for example, leukaemia cells in blood samples have been optically identified using fluorophore-loaded silica beads (Santra et al. 2001). In comparison to traditional staining techniques, nanoparticles provide the benefits of stability and selectivity. For example, quantum dots do not “photobleach” or lose their signal strength over time. Furthermore, populations of nanoparticles of various colours could be conjugated with antibodies directed at various molecular targets. A precise map of the distribution of many molecular markers in a single cell, cell population, or tissue is generated when a single wavelength of light is irradiated (Alivisatos 1996). Protein Detection Proteins are an essential component of the cells’ language, machinery, and structure, and understanding their functions is critical for further advancement in human well-being. Cancer and other disease states are indicated by the presence of certain biomarker proteins and/or abnormal protein concentrations (Ross and Fletcher 1998; Daniels et al. 2004).Another important thing in protein sensing is that it involves binding events. Specific sensors are used on particles surface for recognizing the protein which leads to the generation of fluorescence as output. The quenched complexes are created when the nanoparticles join forces with fluorescent dyes that have complementary charges. The dyes are then displaced by the subsequent binding of protein analytes, generating the fluorescence. Different signal response patterns can then be employed to distinguish the proteins by modifying the connection of the nanoparticle with the protein and/or the nanoparticle with the dye (You et al. 2007).

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However, because of the target analytes’ structural complexity and diversity, protein detection is a difficult task. The enzyme-linked immunosorbent assay (ELISA) is currently the protein detection technique that is most often used (Haab 2006). Although this method possesses high sensitivity the utility is limited due to its high manufacturing costs, instability, and quantification difficulties. The fabrication of materials with adequate surface areas for binding protein exteriors, together with the control of structure and functionality required for selectivity, is the major obstacle for the development of effective protein sensors. Targeting biomacromolecules with sizes comparable to proteins can be accomplished with the help of flexible scaffolds provided by nanoparticles (You et al. 2006). Transducing the binding event is another problem in protein sensing. In immunohistochemistry, gold nanoparticles are commonly used to identify protein–protein interactions. However, this technique’s multiple simultaneous detection capabilities are fairly limited (Tsai et al. 2005). Drug Delivery Nanoparticles as therapeutics can be delivered to specific sites, including those that are difficult to reach with standard drugs. For example, if a therapeutic can be chemically attached to a nanoparticle, radio or magnetic signals can guide it to the site of the disease or infection. These nanodrugs can also be programmed to “release” only when certain molecules are present or when external triggers are present (Tallury et al. 2010). Simultaneously, harmful side effects from powerful medications can be avoided by lowering the effective dosage required to treat the patient. Drug release can be controlled much more precisely than ever before by encapsulating drugs in nano-sized materials (such as organic dendrimers, hollow polymer capsules, and nanoshells) (Yasun et al. 2020). Therapeutic delivery is a very important aspect in the case of healthcare and medicine. Problems associated with free drug such as insolubility in aqueous environment, stability, short half-life, affliction in distribution, and abnormal pharmacokinetic profile can be resolved by using nanocarriers (Din et al. 2017). Drug delivery in a controlled manner has improved therapeutic bioavailability by ignoring unfortunate degradation and enhancing their uptake mechanism. Therapeutic dose can be maintained by controlling the kinetics of release of drugs. DNA nanostructures’ concise addressability enables patterned functionalization with nanoparticles and other protein and hydrophobic groups. This functionalization possesses various applications in the field of biosensing, membrane targeting, and nanofabrication (Patra et al. 2018; Yao et al. 2019). Nanoparticles offer several advantages in therapeutic delivery of drugs: 1. As nanoparticles possess high surface to mass ratio, it can carry a large amount of drug molecules. 2. The size and surface properties of nanoparticles can be altered to accomplish drug targeting. 3. The uptake and internalization with nanoparticles can easily be controlled at targeted site which also reduces the toxicity and other side effects. 4. By selecting appropriate matrix constituents, the rate of drug release as well as the degradation of a carrier can be manipulated.

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5. Furthermore drug entrapment capability is high in the case of nanoparticlemediated delivery and this without any chemical reaction and sustainability of these particles through the physical interactions leads to the preservation of drug activity. 6. The delivery of these drugs is also possible through different pathways like oral, nasal, parietal, intra-ocular, or intravenous. 7. Site-specific targeting can be accomplished by attaching specific ligands to the surface of the nanoparticles (Ferrari 2005). Liposomes are commonly used in drug delivery and as synthetic analogues of cell membranes. To enable lipid membrane binding, DNA has been modified with hydrophobic chemical groups such as tocopherol polypropylene oxide, cholesterol alkyl chains, and porphyrins. Liposome surfaces were improved using cholesterol-modified DNA nanostructures that help in inducing membrane curvature and allow the current flow by forming the membrane spanning nanopores (Franquelim et al. 2018). Recent advances in encapsulation and the development of appropriate animal models have demonstrated that microparticles and nanoparticles can improve immunization (Yang et al. 2016). DNA nanopore dimension is greater than that of natural pore that helps in regulating the ion flow in response to the external stimuli (Burns et al. 2016). It has been demonstrated that the number and location of cholesterol groups on DNA nanostructures influence nanostructure docking and diffusion in lipid bilayers (Mendoza et al. 2017). Nano-formulations protect agents susceptible to degradation or denaturation when exposed to extreme pH, and they also extend drug half-life by increasing formulation retention via bioadhesion. The delivery of antigens for vaccination is another broad application of nanotechnology (Gao et al. 2010; Wang et al. 2018). Gene Delivery Current gene therapy systems are hampered by the inherent difficulties of effective pharmaceutical processing and development, as well as the possibility of an engineered mutant reverting to the wild type. The immunogenicity of viral vectors used in gene delivery is also a concern (Balazs and Godbey 2011). A new age in pharmacotherapy for targeted gene delivery to tissues and cells has arrived with the development of functionalized nanoparticles that can include genetic components such plasmid DNA, RNA, and siRNA with low toxicity (Jin et al. 2009).

6.7

Summary

The properties of nucleic acids make them extremely amenable to their use as constructing materials for nanodevices. Their well-characterized binding patterns via Watson–Crick bonds allow researchers to manipulate the shapes formed by them. Small units of DNA such as the immobile Holliday junction have been used to develop complicated structures which are further utilized as building blocks for larger devices. Additionally, DNA origami has evolved as an.

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essential tool in the fabrication of nanodevices. Various forms of DNA origamibased structures have been developed to design more and more complicated 2D and 3D structures. Beyond the development of nanostructures, DNA has also been used to generate dynamic assembly systems which can perform specific functions. These systems may either require an energy input or may be passive. Recently, various DNA nanorobots such as the nanowalker and DNA switch have been applied to a range of functions. These are discussed briefly. Finally, DNA often needs to be modified via attachment of functional groups which allow them to carry out specific functions. The most common functional groups such as fluorophores and quenchers, photoreactive compounds, hydrophobic moieties, biotin, and redox-active modifications are discussed here. Each of these groups lends additional properties to the nanodevice, and some examples of their use have been discussed. Nucleic acid analogues have also shown increasing popularity for the synthesis of nanodevices. These include PNAs, LNAs, and psDNAs, and these compounds exhibit increased nuclease resistance along with other essential properties. It is expected that the fast growth of this field will soon lead to the generation of devices which will find wide use in robotics, therapeutics, and diagnostics.

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Nucleic Acid in Diagnostics Anindya Dutta, Nilanjan Banerjee, Madhurima Chaudhuri, and Subhrangsu Chatterjee

7.1

Introduction

The Nucleic Acids are the backbone of all living creatures on Earth. These make up the genetic material that constitutes the living embodiment of the most astonishing creation in the Universe, that is Life. Nucleic acids basically are of two types, DNA (deoxyribonucleotide) and RNA (ribonucleotides) that contribute to the precise orchestration of behaviour, motility, growth, repair and metabolism of cells that ultimately establish the living nature of organisms. Although cells within organisms share basically the same universal basic features including cell types, morphology, genetics and reproduction, they differ in their exact genetic sequence makeup of individual entity. Each and every individual ‘being’ is uniquely different in the nucleotide sequence that exists within the cells. This idiosyncratic feature of nucleic acids and the genetic material offers immense potential in the area of diagnostics and prediction of future therapy. The history dates back 2000 years when Hippocrates first predicted the presence of inherited traits. This was followed by revolutionary work by Gregor J. Mendel on the pea plant, gaining recognition after its rediscovery in the 1900s by Hugo de Vries, Carl Correns and Erich von Tschermak. By the beginning of the twentieth century, the genetic material was not known by their names, but their existence had been predicted. Path-breaking research by Albrecht Kossel, Phoebus Levene, Nikolai Koltsov, Frederick Griffith, Oswald Avery, Erwin Chargaff and Alfred Hershey paved the way for unravelling the double helical model of DNA by Francis Crick and James Watson in 1953. With the help of Arthur Kornberg’s discovery of the DNA polymerase, along with the work of Marshall Nirenberg and Har Gobind

A. Dutta · N. Banerjee · M. Chaudhuri · S. Chatterjee (✉) Department of Biophysics, Bose Institute, Kolkata, West Bengal, India e-mail: [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 S. Chatterjee, S. Chattopadhyay (eds.), Nucleic Acid Biology and its Application in Human Diseases, https://doi.org/10.1007/978-981-19-8520-1_7

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Khorana to crack the genetic code, scientists were able to understand the enigma of the ‘code of life’. The turn of the century saw the success of the Human Genome Project (HGP) in 2003, which ushered in the era of detection, identification, synthesis and sequencing of nucleic acids for the development of molecular diagnostics (Anon n.d.). The current millennia have seen the development of sequencing-based technologies like next-generation sequencing (NGS), which has accelerated diagnosis and treatment of fatal diseases (Yohe and Thyagarajan 2017). The use of molecular diagnostics includes the fields of forensics testing (Linacre and Graham 2002), immunotherapy (Sastre and Sastre-Ibañez 2016) and immunosuppression, epigenetics (Sandoval et al. 2013), molecular pathology (Fassan 2018), molecular oncology (Sokolenko and Imyanitov 2018), metagenomics (Chiu and Miller 2019), molecular endocrinology (Rizzo 2021), personalized or precision medicine (Jain 2015) and much more. This provides an immense opportunity to clinicians to moderate the diagnosis, prognosis and monitoring of patients. Nucleic acid testing (NAT) or nucleic acid amplification testing (NAAT) is the detection and amplification of the genetic material of individual organisms. The genetic variation offers the pivotal framework on which the principles of molecular diagnostics are moderated with the knowledge of pharmacogenomics. Molecular diagnostics (Braziel et al. 2003) is mainly used in the detection of infectious (Yu 2012; Okeke and Ihekweazu 2021) and non-infectious diseases (Deshpande and White 2012). Infectious diseases involve the likes of diseases caused by bacteria, viruses, fungi, yeast and parasites while non-infectious diseases include cancer, genetic disorders and mitochondrial disorders (Li et al. 2021a; Maffert et al. 2017). Years of research and understanding in the field of biotechnology and molecular biology facilitated the innovation and development of nucleic acid amplification technologies. The on-going studies focus on catering to the demands of specificity, efficiency and sensitivity with the aim to integrate complex technologies into portable machines and high-throughput analysis. In 1985, Kary Mullis at the Cetus Corporation introduced the polymerase chain reaction (PCR) which has become the ‘gold standard’ in the molecular analysis and amplification of genetic material, having wide range of applications in the fields of detection, identification and characterization of infectious pathogens, genetic diseases, biomarkers, gene expression analysis and preparing samples for further downstream applications. The technique unleashed its potential due to its ability to exponentially amplify minute quantities of DNA molecules which contributed to the increased sensitivity of molecular assays. In the PCR, target DNA is amplified using specific primers that bind to the target region after the duplex double-stranded DNA is separated using heat. The process of replication is carried out by RNA polymerase in cells, in cycles that ensure the exponential amplification of the target DNA region. The amplified DNA is envisaged using gel electrophoresis or other probe-based techniques that allow for their detection by producing colour and fluorescent signals. Addition of a step with the enzyme reverse transcriptase could widen the scope of the technique. RNA is converted to cDNA and PCR amplified to detect minute quantities of RNA from samples like RNA viruses and eukaryotic mRNA. This technique proved useful in detecting diseases even from minuscule amount of genetic material from

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Fig. 7.1 Schematic representation of nucleic acid-based molecular diagnostics

infectious pathogens. Significant progress was made in this field by the introduction of real-time PCR (RT-PCR) technology by Higuchi et. al. which offered higher sensitivity and specificity. The real-time monitoring of the amplification process could be observed by fluorescence-based detectors, increasing the accuracy of quantitation and detection of viral load. Highly automated systems were developed to optimize the detection and precise processing of patient samples in clinical settings. Isolation of nucleic acids from the cells of blood, plasma, serum, cerebrospinal fluid (CSF), sputum, stool and tissues is done using manual or automated methods. Nucleic acids are extracted from the cell and other cellular components using disruption techniques. Chemical lysis includes chaotropic agents, lytic enzymes and detergents, while mechanical lysis encompasses bead-beating, grinding, shearing and shocking. In many cases, one method has been found to be more useful than another. Followed by extraction, NA are amplified and detected using PCR, RT-PCR and sequencing technologies (microarray, NGS, etc.), followed by molecular analysis (Braziel et al. 2003) (Fig. 7.1).

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7.2

Nucleic Acid Techniques in Molecular Diagnostics

7.2.1

Extraction of Nucleic Acids

Nucleic acid extraction propounds the cardinal step in the detection of diseases by molecular diagnostics, routinely used in biological and clinical sciences (Tan and Yiap 2009). It is the starting point of any molecular diagnostic kit, steering research for the development of the process over the years, fostering point-of-care diagnostics in the clinical environment. Extraction involves the process of cell disruption, removal of cellular debris and contaminants, nucleic acid purification and concentration. Nucleic acids commonly occur in the form of plasmids, genomic or chromosomal DNAs and various types of RNAs. Although the structural variations exist between the two forms of nucleic acids, the basic protocol for extraction of DNA and RNA is similar with minor alterations. Commercially available extraction kits work on the guidelines that include rapid, efficient, sensitive, safe, specific and pure isolation of nucleic acids that do not require specialized infrastructure or skills. Disruption involves the interruption of the cell wall or membranes to release the cellular constituents using physical (Goldberg 2008) or chemical methods. The extraction method is dependent on the characteristics of the sample involving a wide variety of tools, often successful either alone or in combinatorial approaches. Depending on the cell type, variations in extraction/cell disruption methods are optimized to meet the desired goal. While mammalian cells are easiest to disrupt, plant cells add another degree of complexity by the presence of a cell wall. Disruption cannot be considered as an isolated technique because it affects all further downstream processes (Haddad et al. 2015). Mechanical techniques (Goldberg 2008) work on the principle of applying sheer stress to extract out the ingredients from the cells using high pressure, ultrasound or excoriation involving rapid agitation by glass or metal beads. Intensive cooling is always necessary to attenuate the heat that was generated by the dissipation of mechanical energy. Bead-beating (Whyte 2016) involves the use of glass or metal beads along with a grinding chamber and agitator, to provide the required kinetic energy for the beads to collide. Diameter and weight of the beads are chosen in accordance with the cell type. Increasing the bead number is directly proportional to degree of disruption due to increased bead–bead interaction. The factors like bead size, number, weight, proportion, cell suspension concentration, agitation speed and agitation design influence the efficiency of disruption, while issues such as poor yield, risk of contamination and generation of heat are of great concern. Glass beads greater than 0.5 mm are used for disruption of yeast cells while size less than 0.5 mm is considered ideal for bacterial and fungal cells. Disruption by ultrasound involves the generation of high frequency vibrations that are transduced into mechanical oscillations through a titanium probe, which is inserted in the cell suspension. Considered ideal for bacterial and fungal cells; bacterial cells require around 30–60 s while yeast cells can take up to 2–10 min to disrupt. Ultrasound-based methods are perfect for the laboratory setting, while scaling up the process generates complexity due to high energy consumption, generation of heat, health and safety issues related to high

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frequency sound. In French press or high-pressure homogenization, the cell suspension is drawn into a cylinder at high pressure and finally released through a small valve. This sudden demise of pressure causes the cells to burst open. It is one of the widely known and used methods for yeast cells and has immense implications in the dairy industry, used for milk homogenization. By maintaining high pressure, the number of passes of the slurry can be modulated to obtain the desired disruption. Factors such as biomass concentration, temperature, number of passes, operating pressure greatly influence the extraction of cell constituents (Costa-Silva et al. 2018).

7.2.1.1 Caesium Chloride/Ethidium Bromide Density Gradient Centrifugation Since the 1950s, the technique of density gradient centrifugation has been widely used in research laboratories. The principle behind the extraction uses the idea that molecules settle down under the influence of a centrifugal force up to the point where the density of the molecules is equivalent to the medium. The differential density of caesium ions and water coupled with interaction with EtBr or propidium diiodide was being used a standard method under laboratory conditions for the separation and purification of various DNA molecules (Fukuda 1976). Under the influence of centrifugal force caesium chloride (CsCl) molecules dissociate; the heavy caesium ions are forced towards the periphery but simultaneously will also diffuse base to the top of the tube, forming a proper density gradient. DNA molecules placed in this medium migrate to different points until the point where it becomes acquainted with the matching density of the medium. This is referred to as neutral buoyancy or isopycnic point and is used for the separation of various types of DNA molecules like chromosomal DNA, plasmid DNA, rDNA or mitochondrial DNA. The density gradient is sufficient for differential separation of varying types of DNA molecules based on the density difference due to GC content and polymorphism (like linear and circular DNA molecules). This coupled with the intercalation of EtBr molecules aids in the successful visualization of separated DNA molecules under the influence of UV radiation. EtBr is an intercalating dye that detects the presence of doublestranded DNA molecules, aiding in the visual detection of supercoiled and non-supercoiled DNA by decreasing the buoyant density of linear molecules. Centrifugation is generally performed at 100,000 rpm (450,000 g) for 10–12 h that require the use of highly expensive ultracentrifuge systems. In a density gradient centrifugation using nuclear DNA performed by Carr and Griffiths (1987) showed the evolution of two bands that corresponded to sheared linear DNA molecules and circular mtDNA. EtBr enhances the visibility of the density difference between the DNA molecules by fluorescence detection. The separated bands appear after centrifugation is collected by making an incision at that point for DNA extraction and subsequent use in various downstream processes (Wright et al. 2009). The main disadvantages involve the higher cost associated with ultracentrifuge machines and the large timeframe required to separate the DNA molecules, making it arduous to perform under the laboratory setting. Moreover, this technique has limited use in the field to clinical microbiology and is impractical for the clinical laboratory (Nasukawa et al. 2017).

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7.2.1.2 Phenol-Chloroform Extraction Phenol-Chloroform extraction exploits the principle of liquid–liquid extraction procedure whereby constituents are separated based on the differential solubility of various substances in two immiscible liquids. Following the use of guanidinium chloride to isolate RNA by Volkin and Carter in 1951 (Volkin and Carter 1951), the work of Grassmann, Defner and Kirby successfully envisaged the efficiency of phenol in the isolation of proteins and nucleic acids from aqueous solutions. The use of guanidinium isothiocyanate was demonstrated by Ullrich et al. and Chirgwin et al. in 1979. Later the work of Chomczynski and Sacchi in 1987 reported the use of guanidinium isothiocyanate along with phenol-chloroform extraction of RNA under acidic environment from tissues rich in ribonucleoproteins like pancreas. This method is used routinely under various laboratory and clinical conditions for isolation of nucleic acids (Silva et al. 2014). This method involves the use of phenol and chloroform followed by vigorous shaking with the sample and centrifugation; this allows phase separation between the upper aqueous phase containing DNA and the lower organic phase containing RNA and proteins. DNA can be precipitated using ethanol or isopropanol at high salt concentration with subsequent washing with 70% ethanol and dissolved in TE buffer or sterile distilled water. RNA is also separated using this method by using guanidinium isothiocyanate followed by phase separation containing RNA in the upper aqueous phase and the lower phase with the DNA and denatured proteins. RNA is precipitated using isopropanol and dissolved in TE buffer or sterile distilled water (Toni et al. 2018). Although this is an easier technique as compared to the CsCl/EtBr density gradient method having widespread utility (Barnett and Larson 2012), its use in the clinical laboratory is limiting due to the toxic, flammable and caustic nature of phenol. 7.2.1.3 Solid-Phase Extraction Liquid-phase extraction is widely used for the isolation of nucleic acids in the clinical and laboratory setting despite having some disadvantages like the requirement of large sample amount, time, resources, laborious methodology, organic solvent and risk of degradation and contamination. With the enhancement of nucleic acid solid-phase extraction methods and microfluidic technology, the use of this technique has gained widespread usage in the field of nucleic acid extraction kits available commercially (Zhao et al. 2018a). The development of nucleic acid-based diagnostic applications like amplification and disease detection, for use inside or outside the scientific laboratory demands the nucleic acid extraction procedures to be fast, economical, high-throughput, simple, automated and highly in sync with further downstream applications. As compared to tradition liquid-phase extraction techniques, solid-phase extraction allows higher purity and yield of the extracted DNA or RNA. This method is based on the principle of solid-phase materials as sorbents of nucleic acids and the accomplishment of ‘binding-and-release’ mechanism of nucleic acids under defined conditions, requiring the use of nucleic acid binding materials for use as sorbents (Li et al. 2022). Boom et al. developed silica-based materials for the separation of nucleic acids and gained widespread attention for its use in commercially available kits (Boom

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et al. 1990). Silica has excellent nucleic acid sorbent capabilities along with rapid release of binding under specific conditions allowing for efficient extraction. Silicabased materials bind to NA using shielded intermolecular electrostatic, hydrogenbonding forces and dehydration effects, having stable chemical properties, lower cost, more bioavailability and modification features (Trachtová et al. 2015). Silica materials (Wang et al. 2020), silica particles (Singh et al. 2019) and diatomaceous earth (Zhao et al. 2018a) are commonly used in SPE methodologies that capture NA in the solid phase in the presence of chaotropic salts followed by washing and elution of NA from silica materials in the presence of lower salt concentration. Studies involving the use of modified surface functionalization of positively charged materials have revealed the higher retention of NA, facilitating improved performance of silica-based materials and diverse applications (Li et al. 2021b). Aminomodified surface functionalization of silica materials proved advantageous for higher binding of negatively charged nucleic acids with positively charged functionalized sorbent materials. Negatively charged carboxylic acid modified magnetic nanoparticles (CAMNPs) are successfully used to isolate single-stranded nucleic acids that had high correlation to the amount of carboxyl surface modifications on nanoparticles (Li et al. 2021b). Magnetic nanoparticles functionalized with hydroxy (HMNPs), amino (ASMNPs) and carboxylic (CAMNPs)-based molecular dynamics simulations revealed the affinity of nucleic acids and various functionalized materials in the order of ASMNPs > HMNPs > CAMNPs. Magnetic nanoparticles conjugated with graphene oxide for extraction of RNA, yielded higher concentration of nucleic acids when compared to traditional procedures using phenol-chloroform extraction (Pham et al. 2017). In 1989, McCormick et al. developed the solid-phase extraction method using siliceous particles resembling the function of phenol; proving advantageous in preventing cross-contamination and DNA loss owing to liquid-phase extraction. Gradually siliceous core particles have been replaced by silica matrix (Esser et al. 2006), glass particles, anion-exchange carriers and diatomaceous earth. This uses centrifugal force to purify DNA through a spin column with ease and efficiency. Most common method involving silica particles, having positive charge and binding to negatively charged DNA molecules enhances efficiency and quantitative approach towards DNA extraction (Close et al. 2016). Boom et al. later introduced an advanced technique involving diatomaceous earth serving as DNA sorbents. The main principle being the immobilization of nucleotide molecules on the matrix in the presence of chaotropic agents. The steps involved can be stated as cell lysis, NA binding to matrix molecules, followed by washing and elution. The column is conditioned using specific buffer of designated pH condition, followed by NA binding and washing the column with reagents that remove contaminants like proteins and salt complexes competitively. Finally, the purified DNA is eluted in TE buffer or sterile distilled water. With the advancement of technology, metal materials have been used as probes, biosensors and NA sorbents. AuNPs (gold nanoparticles) have been widely used for the extraction of ssDNA molecules due to its effective binding propensity to four kinds of deoxynucleotides (Li et al. 2018). The separation of ssDNA by AuNPs is

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dependent on high temperatures, length of DNA molecule and particle size. AuNPs are not commonly used for the extraction of NA since gold is a valuable metal and would increase the cost. Similarly, AgNPs (silver nanoparticles) have been used as NA sorbents, as described by Gu et al. and Yang et al., which revealed that AgNPs bind to N7 of guanine and adenine and N3 of cytosine and thymine. Although AuNPs and AgNPs have not been used for NA isolation, their concomitant use as anti-cancer agents and probe biosensors (Prabowo et al. 2020) using functionalized DNA/RNA for effective and specific targeting of cancer cells and antibodies has attracted much attention for anti-cancer therapeutics. Iron oxide nanoparticles can function as cheap NA sorbents; the superparamagnetism is weakened or destroyed after covalent binding of iron oxide NPs to nucleotides, thus attracting further research which suggested that coating these NPs with silica derivatives conditionally modified with functional groups enhanced the electrostatic binding of NA (SosaAcosta et al. 2020). One important modification for solid-phase extraction technique is the use of magnetic beads for the purification of nucleic acids (Berensmeier 2006). The magnetic beads are negatively charged at the surface that are used to isolate cellular debris and protein from NA. This mitigates the requirement of centrifugation, spin column purification, washing, elution, organic solvents and/or vacuum filtration steps in the solid-phase extraction procedures. Some of the leading companies have coupled the use of silica matrices with magnetic beads for better effectiveness in the purity and isolation time required, focusing on the enhancement of efficiency and convenience in the greater recovery of NA.

7.2.1.4 Applications to Clinical Specimens The extraction of NA from micro-organisms is quite different from that of clinical specimens that are usually under study in the laboratory. The extraction step determines the efficacy and sensitivity of the results and downstream processes involved in molecular diagnostics. The specimens in the clinical laboratory are diverse and are composed of specific characteristics related to its origin, which enhances the need to remove various inhibitors of NA amplification from blood and stool specimens like heme, bile, etc. (Espy et al. 2006). Thus, it requires detailed evaluation and understanding to optimize the extraction method best suited for the isolation of NA from specific sample specimens adapted to one’s need. Examination of routine infectious pathogens from urine (Tang et al. 2005), stool (Machiels et al. 2000), blood (Yamagata et al. 2021), serum (Hoque et al. 2008), cerebrospinal fluid (Casas et al. 1995) is well documented and performed in the clinical setting. Tissues are an important starting point for the evaluation of pathogenic intervention in disease condition by conventional methods like biopsy. These specimens contain cellular debris, proteins, lipids and other NA extraction inhibitors that require an extra purification step to isolate NA from pathogenic strains like herpes simplex virus, varicella zoster, Epstein-Barr virus (Mengelle et al. 2008). Stool is another commonly used specimen for diarrheal diagnosis, which presents a complex approach to remove many unrecognized materials like blood, cellular debris, etc. Blood is one such specimen from which NA are extracted for haematological

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diagnosis of disease-causing pathogens. Many ingredients (like polyanetholesulfonate and hemins) need to be removed from whole blood that inhibit further downstream applications with the extracted NA. Thus, extraction from serum or plasma is quite lucid as compared to whole blood. Extraction of NA from whole blood is outright advantageous because it contained a lot more of NA than serum or plasma aiding convenient quantification. The commercially available kits concentrate on higher recovery rate of NA when heat lysis is coupled with alkaline washing to remove the PCR inhibitors. Investigations have led to the analysis of whole blood specimen using high-throughput automated systems that require less labour and time, being expensive at the same time (Störmer et al. 2007). Dried blood stain is another such approach for isolating minute quantities of NA for congenital infection diagnosis, posing a challenge in being relatively lower in sensitivity due to the insufficient amount of NA present in these samples (Soetens et al. 2008).

7.2.2

Nucleic Acid Amplification Techniques

Molecular diagnostics using nucleic acid testing techniques focus on the detection of miniscule quantities of NAs against the background noise generated by the complex genomic structures. Detecting proper signals is of central importance in nucleic acidbased diagnostics involving clinical specimens. Methods that increase the quantity of target NAs to increase the sensitive detection threshold of NAT are collectively referred to as nucleic acid amplification techniques (NAAT). Amplification techniques of NAs basically consist of target amplification, signal amplification or probe amplification to achieve around million-fold increase in DNA quantity in less time. These amplifications are achieved by cycling or isothermal temperature conditions which are limited by instrument capability and not biochemistry. Amplification can be analysed in endpoint or real-time methods requiring proper positive and negative control setup. Along with PCR, many other methods of amplification have been devised that have found clinical use. These include isothermal amplification methods in which heat denaturation has been replaced by accessory proteins (helicase, recombinase) or strand displacement. These methods still resemble PCR in the products formed. Other methods do not resemble PCR, forming entirely different products based on hairpin extension or the transcription of RNA to DNA.

7.2.2.1 Polymerase Chain Reaction This is the most widely used target amplification technique around the globe in both research and clinical setting, involving the use of thermostable DNA polymerases available, deoxynucleotides of each base (dNTPs), template DNA, and a pair of oligonucleotide primers that are complementary to a specific sequence to amplify the target. The commercial availability of kits, reagents and instrumentation makes it convenient for its wide-scale use in molecular diagnostics. The first step involves the separation of the two strands of DNA duplex by heating them at 95 °C, next the mixture is cooled to an appropriate temperature facilitating the binding of

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oligonucleotide primers to complementary DNA sequences of the template DNA molecule. Subsequently the thermostable DNA polymerase extends the primers at the 3′ end, which are designed in such a way so that DNA amplification extends each strand to include the primer binding site. The thermal cycle is composed of the denaturing temperature (90–97 °C) at which the template and naïve DNA strands separate, while annealing temperature (50–65 °C) is where there is optimal binding of template and primer sequences. DNA synthesis is preferred at temperatures (65–75 °C) between the annealing and denaturing temperatures allowing the extension of DNA strands. The DNA strands are exponentially amplified at the end of the PCR cycle in which each DNA molecule from the previous step is used as the template for subsequent cycles. The instrument used to cycle temperature condition is referred to as thermal cycler. PCR is limited by various inhibitors, primer design, temperature-cycling determinants that influence the efficiency of amplification. Amplification proceeds at an exponential manner at the beginning but plateaus eventually due to saturation conditions generated by the competition between template and primers to bind to one another in the reaction vessel, generating the typical S-shaped logistic curve. With the inclusion of reverse transcription reaction, RNA targets are reverse amplified to their respective cDNA, which appear as templates for subsequent PCR, often involving one-step and two-step PCR. Thermostable enzymes having both DNA polymerase and reverse transcriptase activities facilitate reaction setup in the same tube with the same enzyme. In two-step RT-PCR, the reverse transcription is performed first, usually with random hexamers or a poly-dT oligonucleotide (to prime the poly-A tail of most mRNA). After reverse transcription, the second PCR step is performed on cDNA with specific primers. After amplification, the products can be detected by various methods like the classical method of gel electrophoresis with ethidium bromide staining separating products by size and used for many applications. For zooming down to a single base, one of the primers can be fluorescently labelled and the products separated in conventional sequencers or alternatively, the hybridization assay approaches to verify and analyse the amplified products. Automated closed-tube methods are preferred where the amplified products are not exposed to the outside environment and useful in avoiding contamination in the future with the products of a previous reaction. Adding a fluorescent dye or probe-specific amplification allows real-time detection to follow the reaction progress (real-time PCR) or when the reaction is completed (endpoint melting) without the requirement for a separate analysis step (Rahman et al. 2013).

7.2.2.2 Transcription-Based Amplification Methods These methods are modelled after the replication of retroviruses, known by various names, including transcription-mediated amplification (TMA), NA sequence-based amplification (NASBA) and self-sustained sequence replication (3SR) assays. They amplify their target region in isothermal temperature conditions collectively using the action of reverse transcriptase, RNase H and RNA polymerase and the most widely used is TMA. The primer complementary to the RNA template has a 5′-end which includes a promoter sequence for RNA polymerase, which anneals to the

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target RNA and extended by the enzyme reverse transcriptase, generating RNA-DNA duplex. Next the RNA strand is degraded by RNAse H, allowing the second primer to anneal, and extended by reverse transcriptase generating doublestranded DNA (dsDNA) that includes the promoter. RNA polymerase recognizes the promoter, binds and initiates transcription leading to 100–1000 copies of RNA for one DNA template. Each strand of RNA then anneals and extends the second primer, forming an RNA-DNA hybrid, whereby the RNA in the hybrid is degraded and the promoter primer is extended to produce dsDNA that can be transcribed, repeating the cycle. The reagents are incorporated and amplification proceeds exponentially with reaction completion achieved within an hour. In contrast to PCR, these methods have no requirement for temperature cycling (except for a preliminary heat denaturation for DNA templates). These methods are expedient when RNA targets are used (like human immunodeficiency virus [HIV] and hepatitis C virus [HCV] in blood bank NA screening) (Reta et al. 2020).

7.2.2.3 Loop-Mediated Amplification Methods The loop-mediated amplification procedure was first developed in the 2000s (Notomi et al. 2000) and was generally a preferred method due to higher sensitivity and specificity requiring less time (Nurul Najian et al. 2016). This is a type of isothermal amplification process that uses the strand displacing DNA polymerase from Bacillus stearothermophilus, producing 10 (Rizzo 2021) copies of DNA from minimal starting material within an hour between 60 and 65 °C temperature condition without the requirement of thermal cycling. This technique requires four different primers (extended to six for faster amplification) that recognize six specific sequences within the DNA molecule (backward inner primer (BIP), forward inner primer (FIP), backward and forward loop primers for faster amplification, and the backward outer and forward outer primers). As opposed to conventional amplification techniques like the PCR, loop-mediated amplification (LAMP) generates an array of DNA molecules having branches and loops. There are basically two strand displacements and two loop-formation primers which recognize six segments specific to the target, where both the inner primers include a 5′-tail which is complementary to the target sequence. When the extension of the inner primers is completed, hairpins or loops are formed at each end, where one of them contains a free 3′-OH end that can extend further. This scenario is quite like the snapback and self-probing primers apart from the fact that the 3′-end is unblocked. The displacement of the inner extended region by the outer primers provides the initiation point for cyclic amplification. The chain reaction is started by the extension of the 3′-ends including additional priming by the inner primers which binds to single-stranded regions that are exposed within the loops. The amplification proceeds by the production of complex products with more and more loops and branching structures (Tomita et al. 2008). LAMP has been incorporated in detection, testing and assays for medical investigation. This technique having a detection limit of 1 ng has been used in the identification of resistance genes (Solanki et al. 2013) and detection of pathogens like E. coli (Stratakos et al. 2017), Klebsiella pneumoniae, Mycobacteria, Vibrio parahaemolyticus (Liu et al. 2017), Salmonella sp. (Liu et al. 2017),

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Acinetobacter baumannii, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans. The advantages of LAMP include the non-requirement of initial denaturation step and thermal cycling conditions, lower inhibition by substances present in biological samples and easy detection methods which has favoured LAMP in poor clinical setting environment and point-of-care diagnostics.

7.2.2.4 Helicase-Dependent Amplification (HDA) Helicase-dependent amplification is another type of isothermal nucleic acid amplification procedure that uses DNA helicase to unwind and separate the DNA duplex without the need for thermal cycling conditions. The reaction proceeds with the specific extension of target sequence using two oligonucleotide primer sequences. The helicase used in HDA is a thermostable one, belonging to the superfamily II helicases that is obtained from Thermoanaerobacter tengcongensis (TteUvrD), which is proficient at unwinding nucleic acid substrates at temperatures of 60–65 ° C. When coupled to reverse transcription reaction, HDA has been fruitful for amplifying RNA substrates. This type of amplification technique requires the precise orchestration between DNA polymerase, DNA helicase and single-stranded DNA binding protein (SSB). The DNA undergoes a type of ‘thermal breathing’, whereby the regions rich in adenine and thymidine partially open up to form ‘bubbles’. These are the sites onto which DNA helicase can dock and move along the template molecule to unwind the DNA, facilitating the primers to hybridize to complementary sequences on the template. This is followed by exponential amplification by DNA polymerase to generate new strands of nucleic acids (Cao et al. 2013). The most important aspect of HAD is the optimized formulation of polymerase, helicase and SSB protein that are made available in the market by BioHelix and New England BioLabs as ready-made kits. The products are generally visualized by agarose gel electrophoresis, fluorescent reporter dyes (like SYBR green), fluorescent probes or may be detected at the end point by using an amplicon containment device embedded with a flow strip. Detection with fluorescent reporter dyes usually requires an isothermal fluorimeter, which is similar to real-time PCR, to detect product formation in real time (isothermal fluorimeter developed by Qiagen). Since this process does not require thermal cycling conditions, it has immense potential for development of portable DNA point-of-care diagnostic devices (Barreda-García et al. 2018). 7.2.2.5 Signal-Mediated Amplification of RNA Technology (SMART) The SMART technique revolves around the detection of amplified signal rather than the amplified target, thereby not requiring the use of thermal cycling conditions and target sequence copying is not necessary. The signals are generated based on specificity, unique for various targets, and can distinguish between different targets by detecting changes in base-pairs. This includes a single-stranded oligonucleotide called ‘extension probe’, for extending the sequences and another single-stranded DNA serves as the ‘template probe’. SMART is an isothermal nucleic acid amplification technology that uses a three-way junction (3WJ) structure to generate copies of an RNA product. Both the probes comprise a longer end which binds to the target, and a shorter end which is used to hybridize with each other, producing a 3WJ

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structure only in the presence of the target sequence. The template probe contains a single-stranded non-functional T7 RNA polymerase promoter sequence. Two enzymes are mainly involved in this reaction, a T7 RNA polymerase and Bacillus stearothermophilus DNA polymerase which can function under the same reaction setup. When 3WJ structure is formed and DNA polymerase is added to the reaction mix, it extends the ‘extension probe’ along the ‘template probe’, to produce a functional double-stranded T7 RNA polymerase promoter sequence, leading to the generation of RNA signal. The signal is amplified when the generated RNA signal hybridizes to another template oligonucleotide, which is further extended by DNA polymerase to produce enhancement in signal production due to double-stranded promoter sequence formation. Further rounds of extension and transcription lead to increase in RNA yield which can be detected by a method called enzyme-linked oligosorbent assay (ELOSA). An enzymelinked oligonucleotide and another biotin labelled oligo binds to RNA signals with sequence specificity, forming a complex that is captured in streptavidin coated wells, visualized by measuring the colour changes that are observed upon addition of substrate. The sensitivity of SMART is lower than that of PCR, with detection limit ranging from 50 nmol single-stranded synthetic target, 10 ng genomic DNA and 0.1 ng total RNA from bacteria. This technique has been applied in the detection and analysis of marine phage viruses of Synechococcus, gene expression analysis for detecting perturbation between diseased and healthy hosts and uncovering of genes of MRSA (Hall et al. 2002).

7.2.2.6 Nucleic Acid Signal-Based Amplification (NASBA) In contrast to SMART, NASBA encompasses the actual amplification of ssRNA and DNA. Detection of RNA by RT-PCR has limitations due to cross-contamination and presence of false positives; NASBA is a one-step isothermal amplification technique that encompasses methods such as transcription-mediated amplification and selfsustained sequence replication (3SR) (Compton 1991). This is applied for the amplification of genomic RNA, messenger RNA, ribosomal RNA and cannot be used for amplifying dsDNA unless they are denatured initially (Li and Macdonald 2015; Zanoli and Spoto 2012). NASBA has no requirement of specialized instrumentation and can amplify RNA targets up to one million-fold (three to four cycles) in only 90 min approximately (Hønsvall and Robertson 2017). NASBA involves three thermolabile enzymes, T7 DNA-dependent RNA polymerase (DdRp), RNaseH and reverse transcriptase from avian myeloblastosis virus (AMV) requiring two primers. The initial denaturation is done at a different temperature (65 °C for RNA and 95 °C for DNA) than the actual amplification which is carried out at 41 °C. The amplified products are analysed by gel electrophoresis, electrochemiluminescence (ECL) or enzyme-linked gel assay (ELGA). NASBA is limited by the use of thermolabile enzymes after the denaturation steps and can only amplify targets which are 120–250 bp in length. It also has several advantages like lower operating temperature (41 °C) and time, less contamination than convention PCR method, faster amplification kinetics and less dependency on primers to affect amplicon yield (Rutjes Saskia et al. 2006; Cordray and RichardsKortum 2012). It is used for the diagnosis of RNA viruses and NASBA-linked

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molecular beacons have been developed for real-time detection of RNA. Molecular beacons are oligonucleotide probes that contain a fluorescent tag at the 5′-end and a quencher at the 3′-end. These are highly target-specific probes that anneal to the amplified target RNA in the NASBA amplification reaction facilitating quantitative real-time detection (Leone et al. 1998). One of the primers (P1) binds to RNA target and is extended by AMV reverse transcriptase enzyme. RNaseH destroys the RNA strand of the RNA-DNA hybrid; another primer P2 binds to the target which is elongated by AMV reverse transcriptase to produce a double-stranded DNA molecule. The design of P1 is such that it aids in the production of a T7 RNA polymerase promoter site which is used to generate anti-sense RNA copies from DNA template. The same is done for the sense strand, where the second primer (P2) binds first and produces new copies of DNA from RNA. Thus, DNA is produced as the end product which contains a promoter region, allowing T7 RNA polymerase to use it as a template. Although the reaction sequences from the sense and anti-sense strands are different, they are deemed kinetically the same, referred to as copy DNA (cDNA). Over the years, various improvements have been made to the NASBA technology for use in a wide scope of clinical applications. Several kits have been developed for quantitating HIV-1 in human clinical samples: for example, NucliSENS EasyQ System for detection and quantitation of HIV-1 viral RNA. Quantitative detection by NASBA (NASBA-QT) has been successfully used for quantitative detection of human papillomavirus (Zappacosta et al. 2017), rhinovirus (Loens et al. 2003), human cytomegalovirus in HIV-1 patients (Zhang et al. 2000), HCV (Damen et al. 1999), hepatitis A (Jean et al. 2001), Leishmania parasite (van der Meide Wendy et al. 2005).

7.2.2.7 Recombinase Polymerase Amplification (RPA) This is an isothermal amplification method for nucleic acids that utilizes recombinase and polymerase enzymes to operate at lower temperatures (37–42 ° C). The amplification reaction takes place in about 20–40 min and is one of the fastest NAATs available in the market for detection and diagnosis of human pathogens, with detection limit as low a one copy of target. The RPA reaction begins with the binding of recombinase enzyme (RecA from E. coli) with the primers, in the presence of a molecular crowding agent (high molecular weight PEG), to form a recombinase-primer complex. This complex then scans the entire target for homology, and once found, the entire complex promotes strand displacement to form a D-loop structure. This D-loop structure is stabilized by singlestranded binding proteins (SSBs), which enhances primer-target hybridization. Once the disassembly is achieved by the recombinase complex, the DNA polymerase (Bacillus subtilis Pol I or Sau recombinase polymerase) with strand displacement activity binds to the 3′-end of the primer and extends it (Lobato and O’Sullivan 2018). Improvement in technology has provided greater sensitivity, cost effectiveness, less manual handling and errors, which proved to be appropriate for rapid molecular diagnosis. The RPA-based methods have equivalent sensitivities with that of the

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conventional PCR techniques but with much less time. The initial denaturation step is also not required, which abrogates the need for thermal cycling instrumentation. The amplification reaction can proceed easily in the presence of high concentration of PCR inhibitors. Different types of RPAs have been developed over the years, like the digital RPA, RT-RPA and multiplex RPA for the detection of pathogens (Li et al. 2019). Multiplex RPA has been used for the detection of Neisseria gonorrhoeae, Salmonella enterica and Staphylococcus aureus. Scientists have been able to detect as low as 10 copies of target DNA of HIV-1 in 15 min using RPA (with a lateral flow assay) for HIV-1 diagnostics in resource-poor environment. RPA has also been used for the detection of tuberculosis in 20 min from femtogram quantity of template DNA. RPA technology has been translated to the laboratory and clinical setting, with available commercial kits and reagents for both endpoint and real-time detection. A FRET-based fluorescent probe and lateral flow strip scheme have been incorporated in the RPA method for the detection of SARS-CoV2 N gene quite easily with diminished background noise levels. These examples prove the high efficacy of RPA in point-of-care diagnostics, detection of biomarkers, food and agricultural industries (Daher et al. 2016).

7.2.2.8 Rolling Circle Amplification (RCA) The rolling circle amplification method has been developed from rolling circle mode of DNA replication observed in vivo. It has a lower operating temperature (23–60 ° C) and requires a DNA polymerase having strand displacement property (Phi29 DNA polymerase), which uses circular template (like plasmids, nucleic acids from virus/bacteria) and a specific primer to produce long DNA molecule having tandem repeats. When using linear DNA molecule, an intermediate circular template is generated which aids in subsequent RCA reaction. This method has high amplification efficiency which is enhanced in the presence of single-stranded DNA binding proteins (SSBs) (Bhat and Rao 2020). Several years of research have seen the development of different RCA-based techniques for exponential amplification of target molecule, such as ramification amplification (RAM), hyperbranched RCA (HRCA), cascade RCA and multiply-primed RCA. The property of this method to produce long amplified fragments has great value in whole-genome amplification, during the analysis of viral DNA genomes. RCA can also be applied for use on solid support, inside cells or cell surfaces for enhanced molecular detection. RCA-based kits are available in the commercial market, which are employed in the research setting for laboratory use and molecular diagnostics (Xu et al. 2021; Goo and Kim 2016). 7.2.2.9 Strand Displacement Amplification (SDA) The strand displacement amplification (SDA) technique was developed by Walker et al. in 1992, which employs an endonuclease, a DNA polymerase without exonuclease activity and two primer sets. First set of primers contain restriction site 5′-overhangs, with specific recognition site for HincII restriction enzyme and another set called ‘bumper primers’, which assist the displacement of amplified product from the first primer set. DNA is denatured by heat and the first set of

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primers hybridizes to the target. Due to the use of thiol-modified dATP, the original primer is cleaved by HincII and not the newly synthesized strand. This results in 3′-overhangs which is extended by exonuclease-deficient DNA polymerase (exo-klenow), which additionally displaces the downstream strand. Coupling of sense and anti-sense reaction results in exponential amplification, where the displaced anti-sense strand serves as a template for the sense-strand reaction and vice versa (Hellyer and Nadeau 2004). This isothermal technique with low operating temperatures (30–55 °C) is prone to non-specific amplification due to primers binding at unspecific site, which prevents the development of diagnostic application using SDA. This is exaggerated in clinical samples, since the DNA/RNA extracted from them contains a large number of similar target sequences (homologous loci/ gene families) which might miss-prime the target sequence. SDA has high amplification efficiency for short sequences (50–120 bp) and has focused on the miRNA expression profiling. Another disadvantage of this technique is the requirement of large amount of time (2.5 h) which makes it difficult for on-site detection. SDA has been used for clinical diagnosis of Chlamydia trachomatis, Neisseria gonorrhoeae and herpes simplex virus from urogenital samples, on a high-throughput platform (Suther et al. 2022).

7.3

Nucleic Acid Testing for the Detection of Diseases

7.3.1

HIV-I

HIV results in immunodeficiency syndrome (AIDS), primarily affecting the human immune system, also creating a significant risk of opportunistic infections and malignancies. As of 2020, 37.7 million [30.2 million–45.1 million] were infected globally, with 1 million people dying from AIDS-related diseases. Detection and treatment are crucial in averting an AIDS outbreak on a broad scale. Laboratory and clinical microbiologists use a variety of tests to diagnose a patient’s HIV infection status, assess disease progression and track the success of antiretroviral medication (ART). Variety of methods like virion visualization in cell culture, assessment of HIV-specific antibody and viral antigens and detection of viral nucleic acids can all be used to screen for HIV infection. For testing the presence of HIV antibodies in conventional HIV assay, there exists a window during which a person will have negative test results for HIV antibody despite the presence of infection, this is called eclipse phase. The eclipse phase is defined as the time between HIV infection and a diagnosable infection. A nucleic acid test (NAT) can typically detect HIV infection 10–33 days after an exposure. It takes around 18–45 days after an exposure to detect HIV antigen/antibody from blood taken from vein by a laboratory (Fig. 7.2). HIV antigen/antibody tests done with finger prick blood can take longer time to detect (18–90 days after an exposure). Most rapid tests and self-test kit are antibody tests (Table 7.1). In comparison with antibody testing, the projected mean window-period reduction for HIV-1 RNA by pooled sample NAT is around 11–15 days.

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Fig. 7.2 Virological and serological markers following infection with HIV

Two frequent qualities required for molecular instruments aiming at HIV detection or diagnosis are the availability of data in a fairly short amount of time and a robust standardization of the extraction and amplification stages. Two critical components of integrated NAAT equipment are (1) efficient nucleic acid extraction technologies for different and complicated sample types and (2) robust and sensitive nucleic acid proliferation and detection technologies. Since 2014, an additional HIV-1/HIV-2 differentiation test has been introduced in the approved laboratory testing protocol for diagnosing human immunodeficiency virus (HIV) infection to confirm infection type based on the presence of type-specific antibodies. Because the epidemiology and clinical management of HIV-1 and HIV-2 infections varies, it’s critical to distinguish between the two. The industry invested much in the establishment of NAT for assessing human blood and blood component donors. Because of the high cost and labour intensity of inspecting plasma donor samples, NAT received a lot of attention in testing clusters of plasma donor samples. Initially to establish NAT as the best detection method large-scale clinical studies were performed and in the year 1999 FDA first published a guide on test standards, manufacturing requirements and clinical trial requirements for industry on the

99.40%

98.48%

99.70%

100.00%

100.00%

100.00%

Determine HIV Early Detect (former Alere HIV Combo)

Rapid Test for Antibody to Human Immunodeficiency Virus (HIV) (Colloidal Gold Device) INSTI HIV-1/HIV2 Antibody Test Kit

Genie Fast HIV 1/2 99.00%

99.40%

100.00%

100.00%

Final specificity 99.70%

Product name ABON™ HIV 1/2/O Tri-Line Human Immunodeficiency Virus Rapid Test Device Determine™ HIV-1/2

Initial sensitivity 100.00%

BioLytical Laboratories, Richmond, Canada Bio-Rad Laboratories, Marnes La Coquette France

Beijing Wantai Biological Pharmacy Enterprise Co.

Manufacturer ABON Biopharm (Hangzhou) Co. Ltd. Hangzhou, PR China Abbott Diagnostic Medical Co. Ltd, Matsudo, Japan Abbott Diagnostic Medical Co. Ltd, Matsudo, Japan

18 months 2–30 °C

15 months 15–30 °C 18 months 2–30 °C

Serum/plasma/ whole blood

Serum/plasma/ whole blood Serum/plasma/ venous and capillary whole blood

HIV 1/2 antibodies combined detection HIV 1/2 antibodies (group M and O)

18 months 2–30 °C

Serum/plasma/ whole blood

Discrimination between HIV 1/2 antibodies combined detection and HIV1p24 antigen HIV 1/2 antibodies combined detection

18 months 2–30 °C

Serum/plasma/ whole blood

Specimen type Serum/plasma/ whole blood

Anticipated shelf life (months)/ storage temperature 24 months 2–30 °C

HIV 1/2 antibodies combined detection

Analyte Discrimination between HIV-1 and HIV-2 antibodies

Table 7.1 List of HIV Diagnostic test kits classified according to the Global Fund Quality Assurance Policy prequalified by WHOa

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a

100%

99.8% HIV-1 (fingerstick whole blood) 99.9% HIV-1 (venous whole blood, serum, plasma) 98.9% HIV-1 (oral fluid) 100% HIV-2 (serum/plasma, blood, oral fluid) 100%

100%

99.20%

99.9% (serum/ plasma, whole blood, oral fluid)

OraSure Technologies Bethlehem, USA (manufactured in Thailand) Premier Medical Corporation Private Limited, GIDC, Gujarat, India

Report taken according to Global Fund Quality Assurance Policy for Diagnostic Products

First Response® HIV 1-2-0 Card Test (version 2.0)

OraQuick® HIV-1/2Rapid Antibody Test

DPP HIV 1/2 Assay

and Steenvoorde, France Chembio Diagnostic Systems, Medford, USA

Discrimination between HIV-1 and HIV-2 antibodies

HIV 1/2 antibodies combined detection

HIV 1/2 antibodies combined detection

Serum/plasma/ whole blood

Serum/plasma/ whole blood/oral fluid

Serum/plasma/ venous whole blood/fingerstick whole blood/oral fluid

24 months 4–30 °C

30 months 2–30 °C

24 months 2–30 °C

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evaluation of NAT plasma donor screening procedures. The first NAT system, the National Genetics Institute (NGI) UltraQual HIV-1 and HCV Reverse Transcription Polymerase Chain Reaction (RT-PCR) assays, to perform RT-PCR assays on pooled samples from donors of Source Plasma was licensed in September 2001. Subsequently next year FDA licensed two NAT, Procleix HIV-1/HCV Assay, a qualitative NAT for detection of HIV-1 RNA and COBAS AmpliScreen HIV-1 Test, v 1.5, a qualitative in vitro test for the direct detection of HIV-1 RNA in plasma samples from human donors, including donors of whole blood, blood components, and source plasma, and from other living donors, in February and September, respectively. Currently there are more than 20 different HIV detection NAT kits. Some of them are mentioned in Table 7.2. Lucia Hans further demonstrated that cobas (cobas HIV-1/2 Qual) HIV-1/HIV-2 qualitative test on cobas 6800/8800 Systems facilitates early and precise diagnoses of HIV-1 and HIV-2 in adults and children across sample types (Hans et al. 2021). They concluded that this assay could lead to survival by blocking the transmission of acute HIV and simplify the HIV diagnostics algorithm. Adams et al. gauged the performance of the cobas® HIV-1 quantitative NAT on the cobas 4800 HIV-1 system and exhibited 100% specificity. They concluded that cobas system is highly sensitive, accurate and correlates well with other assays (Adams et al. 2019). In October 2004, FDA issued a final guidance, ‘Use of Nucleic Acid Tests on Pooled and Individual Samples from Donors of Whole Blood and Blood Components (including Source Plasma and Source Leukocytes) to Adequately and Appropriately Reduce the Risk of Transmission of HIV-1 and HCV (October 2004 guidance) and informed organizations amassing blood and blood components to use licensed NAT that meet the criteria in 21 CFR 610.40(b) to screen blood donors for HIV-1 RNA. Morris et al. tested 3151 persons, 79 had newly diagnosed cases of HIV: 64 had positive results from rapid HIV test, and 15 had positive results only by NAT (that is, NAT increased the HIV detection yield by 23%) (Morris et al. 2010). Due to early detection, NAT reduces transmission during acute HIV. Study by Chang et al. using Alere q HIV-1/2 Detect test (Alere Detect), a rapid point-of-care (POC) nucleic acid test (NAT) that identifies and discriminates HIV-1 and HIV-2 in 25-μL whole blood or plasma samples, revealed that it has the potential for use as a rapid HIV-2 NAT-based diagnosis tool in resource-limited settings. The Alere Detect was able to accurately discriminate HIV-1 and HIV-2 in 100% patient samples with detectable HIV RNA (Chang et al. 2017). It might be difficult to cope with an HIV diagnosis. However, today’s HIV patients have a wide range of therapy alternatives. Study by Marcus et al. suggests that adults with HIV infection may have a life expectancy similar to that of people who are HIV negative. However, further awareness is necessary to avert comorbidities among HIV-positive people (Marcus et al. 2020). A data from 2017 by WHO shows that in the European Union/European Economic Area nearly 90% of AIDS diagnoses occurred within just 90 days of the HIV diagnosis. This implies that majority of these cases could have been averted with initial diagnosis. (Lockdowns due to COVID-19 pandemic and reduced transmission due to physical distancing in

N/A

N/A

N/A

N/A

N/A

N/A

N/A

Xpert HIV-1 Viral Load

Xpert HIV-1 Qual Assay

SAMBA HIV-1 Semi-Q

COBAS AmpliPrep/COBAS Taqman HIV-1 Test Version 2.0 (Taqman 48) COBAS® AmpliPrep/ COBAS®TaqMan® HIV-1

N/A

N/A

N/A

N/A

N/A

NucliSENS EasyQ HIV-1 V2.0 (Automated)

N/A

Final specificity N/A

N/A

Initial sensitivity N/A

m-PIMA HIV-1/2 Detect

Product name Abbott Real-Time HIV-1 (Manual)

Roche Molecular System, Branchburg, USA

Roche Molecular System, Branchburg, USA

Diagnostics for the Real World, Sunnyvale, CA 94085, USA

Cepheid Inc., Rontgenvagen 5SE-171, 54 Solna, Sweden

Cepheid Inc., Rontgenvagen 5SE-171, 54 Solna, Sweden

Abbott Rapid Diagnostics Jena GmbH, Germany Loebstedter Str. 103-105 07749 Jena, Germany bioMerieux SA, Marcy l’Etoile, France

Manufacturer Abbott Molecular Inc., Des Plaines IL, USA

HIV-1 Quantitative NA target HIV-1 Qualitative NA target HIV-1 SemiQuantitative RNA HIV1 Quantitative RNA

HIV1 Quantitative RNA

Detection type HIV 1 Quantitative RNA HIV-1/ 2 Qualitative RNA

24 months 2–30 °C

Nucleic Acid in Diagnostics (continued)

Plasma or PSC dried plasma spot (with PCS) Plasma or dried blood spots

Plasma

9 months 2–37 °C 18–24 months 2–30 °C

Whole blood and DBS

12 months (2–28 °C)

18 months (2–28 °C)

Plasma dried blood spot (venous whole blood) Plasma

Whole blood, plasma

9 months (4–30 ° C)

15–24 months (2–30 °C)

Specimen type Plasma

Anticipated shelf life (months)/ storage temperature 18 months

Table 7.2 List of HIV NAAT diagnostic test kits classified according to the Global Fund Quality Assurance Policy prequalified by WHOa

7 233

a

Initial sensitivity

Final specificity Manufacturer

Report taken according to Global Fund Quality Assurance Policy for Diagnostic Products

Qualitative Test, version 2.0 (TaqMan 48)

Product name

Table 7.2 (continued)

Detection type HIV1 DNA & RNA Qualitative

Anticipated shelf life (months)/ storage temperature Specimen type

234 A. Dutta et al.

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the year 2020–2021 made the interpretation of HIV trends for 2020 challenging and debatable.) Early detection of HIV can be a boon for the society in which NAT plays an exceptional role.

7.3.2

Cancer

Earlier cancer research was solely focused on 1–2% coding genes in the human genome and particularly concentrated on genetic mutation and/or modulated protein structure/function. Currently scientists are shifting their focus to 98–99% non-coding transcripts of the human genome. As a result, several non-coding RNAs and their functional processes in most of the cancer have been discovered and characterized (Chan and Tay 2018). The alterations of genomic DNA and RNA also deliver latest acumen into the functional mechanisms of nucleic acids in cancer (Liao et al. 2020; Pekarsky and Croce 2019). Transcription control is more complex than traditional regulation by transcription factor as various kinds of DNA modification lead to the changes in the expression pattern of oncogenes which leads to cancer. Further, the functional activity of coding and non-coding RNAs is tweaked by RNA modifications. These has led to the establishment of the genetic material as the main driving force for cancer. Nucleic acids also act as biomarker for cancer. Fragmented DNA and RNA indicated as circulating nucleic acids are present inside the cancer cell as well in the extracellular region like blood and other body fluids like plasma, serum, urine, saliva and milk (Sohel 2020; Huang and Yu 2015; Brunner et al. 2012). According to recent research, ectopic lncRNA expression is implicated promising in angiogenesis, cell proliferation, migration and apoptosis, and hence is a key component of carcinogenesis (Prensner et al. 2011; Jana et al. 2017).. Several of lncRNA have been proved to act as promising non-invasive cancer biomarkers due to their elevated presence in cancer patients (Bartonicek et al. 2016; Bolha et al. 2017) (Table 7.3). MicroRNAs (miRNAs), another class of 18–22 nucleotide non-coding RNA molecules, are involved in so many developmental processes. Since they impact post-transcriptional gene expression by destabilizing mRNA and/or hindering translation it’s expected that variations in their expression are linked to abnormal circumstances like ill-developed embryo, poor growth, cancer development, progression and dissemination (Tornesello et al. 2020). Several studies revealed that these miRNAs and lncRNAs can be detected in the serum and other biological fluids. A study by Md Mahmodul Hasan Sohel revealed several miRNAs having the potential to act as biomarker (Table 7.4). These cancer-associated circulating nucleic acids can be easily detected in body fluids and can be used in the estimation of cancers between characteristic tumour patients from healthy people at early phases with both high sensitivity and specificity. Thanks to the nucleic acid detection techniques one can now easily diagnose cancer. NAT identifies changes in gene sequence as well as copy number and can be used to distinguish entities who are prone to have a recurrence and who are likely to respond to a specific treatment, as well as to distinguish between different tissue

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Table 7.3 Cancer-associated lncRNAs in cancer cells with biomarker potential Type of cancer Glioma

Regulation type Upregulation Upregulation Upregulation Upregulation

Fold changea 2.0–5.0 5.0–10.0 >2.6 2.0–6.0

HOTAIR ATB MALAT1 CCAT2 ANRIL LSINCT5 LINC00617 RP11445H22.4 GAS5

Upregulation Upregulation Downregulation Upregulation Upregulation Upregulation Upregulation Upregulation

5.2 >40.0 3.3 7.5 >1.5 2.0–7.0 >1.5 15.0–20.0

Downregulation

2.0 >1.0 32.7 >1.0 >2.0 >1.0 32.9 >2.0

Prostate

PCAT18

Upregulation

8.8–11.1

Colorectal

Lung

Breast

Hepatocellular

Oesophageal Bladder

Name of lncRNA MALAT1 ATB Linc-POU3F3 MALAT1

References Yu (2012) Mansoori et al. (2017) Guo et al. (2015) Vallabhapurapu et al. (2015) Svoboda et al. (2014) Huang et al. (2016) Weber et al. (2013) Qiu et al. (2014) Nie et al. (2015) Silva et al. (2011) Li et al. (2017) Xu et al. (2015) Mourtada-Maarabouni et al. (2009) Tang et al. (2015) Tang et al. (2015) Tang et al. (2015) Shi et al. (2015) Geng et al. (2011) Huang et al. (2015) Panzitt et al. (2007) Luo et al. (2016) Li et al. (2014) Zhu et al. (2015) Srivastava et al. (2014) Mazar et al. (2014), Khaitan et al. (2011) Crea et al. (2014)

cancers. These variations can often be used to anticipate how the cancer will behave in the upcoming days, including if it will spread and how it will respond to different treatments. Oncologists and pathologists are increasingly relying on NAT to aid diagnosis and treatment planning. Genetic factors like inherited mutations can drive the onset of cancers. For example, mutation in gene TP53 causes Li-Fraumeni syndrome that leads to high risk of developing cancer (Kratz et al. 2021); BRCA1 and BRCA2 genes are related with hereditary breast and ovarian cancer (Tornesello et al. 2020; Schrijver et al. 2021); PTEN mutation causes Cowden syndrome and boosts the risk of thyroid,

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Table 7.4 Biomarker potential of upregulated circulating miRNAs in cancer Type of cancer Lungs

Gastric

Breast

Colorectal

Pancreatic

Name of miRNA miRs-21 and miR-210

Sample type Plasma

miR-210-3p

Serum

miR-31

Serum

miR-21

Plasma

miR-21-5p, miR-2233p, and miR-9-5p

Serum

miR-221, miR-744, and miR-376c Yes miR-196a

Serum

miR-106a, miR-106b, miR-21, and miR-93 miR-19b-3p and miR-106a-5p miR-21-3p and miR-21-5p miR-188-3p, miR-500a-5p and miR-501-5p miR-17-5p

Plasma Plasma Serum exosome Serum Serum exosome Serum

miR-5698 and miR-8089

Serum

miR-141

Plasma

miR-17-5p, miR-18a5p, miR-181a-5p and miR-18b-5p miR-338-5p

Plasma exosome Serum

miR-10b, miR-21, miR-30c, and miR-181a miR-182

Plasma and exosome Plasma

miR-21-5p

Plasma Serum

Sensitivity and specificity 75.0% and 84.8% 86% and 79% 76.9% and 74.5% 82.1% and 96.4% 82.69% and 88.00% 82.4% and 58.8% 69.5% and 97.6% 7 84.8% and 79.2% 95% and 90% 97.9% and 73.5% Not studied

Biomarker potential Yes Yes Yes Yes Yes

Yes Yes Yes Yes Yes Yes

100% and 75.4% 69% and 76%

Yes

77.1% and 89.7% 0.769 and 0.867

Yes

85% and 88.8% 100% and 100%

Yes

64.1% and 82.6% 85% and 100% 95.3% and 76.7%

Yes

Yes

Yes

Yes

Yes Yes

Reference Liu et al. (2016) Świtlik et al. (2019) Yan et al. (2015) Sun et al. (2018) Yang et al. (2018) Song et al. (2012) Tsai et al. (2016) Zhao et al. (2018b) Wang et al. (2017a) Yu et al. (2018) Zou et al. (2020) Swellam et al. (2019) SatomiTsushita et al. (2019) Cheng et al. (2011) Zhang et al. (2019) Bilegsaikhan et al. (2018) Lai et al. (2017) Chen et al. (2014) Karasek et al. (2018) Zou et al. (2019) (continued)

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Table 7.4 (continued) Type of cancer

Name of miRNA let-7b-5p, miR-19a-3p, miR-19b-3p, miR-253p miR-192-5p, and miR-223-3p miR-221-3p

Sample type

Plasma

Sensitivity and specificity

76.3% and 63.6%

Biomarker potential

Yes

Reference

Calabrese et al. (2018)

breast and other cancers (Yehia et al. 2020). NAT test like DNA sequencing can easily detect which person has inherited these mutations and prevent oncogenesis. Compared to conventional single-gene and array-based methodologies, sequencing employing next-generation sequencing (NGS) methods gives more detailed information in short time. A major advantage of NGS is it can analyse multiple mutations and sequences from the same sample to detect rare somatic variations, tumour subclones and circulating DNA fragments. Non-small-cell lung cancer (NSCLC) is generally caused by rearrangement/translocation of anaplastic lymphoma kinase (ALK), epidermal growth factor receptor (EGFR) and receptor tyrosine kinase (ROS1), hence analysis of these genes has already been incorporated in the NSCLC diagnostic standards (Dietel et al. 2016; Imyanitov et al. 2021) (Table 7.5). In present clinical practice, tissue diagnosis for cancer biomarker is still judged to be the gold standard for initial diagnosis of NSCLC. A study of 282 patients with formerly untreated NSCLC proved that there was a 48% increase in the rate of biomarker detection with circulating tumour DNA testing compared to tissue analysis. The Guardant360 and the CDx assay Foundation One Liquid CDx are FDA-approved comprehensive pan-tumour liquid biopsy test. Both identify circulating NA from the blood. Treatment options for late-stage malignancies are frequently limited. Five-year survival rates for certain cancers like colon cancer are as high as 92%, provided they are detected at stage I. In the Taizhou Longitudinal Study (TZL), ~120,000 healthy subjects provided plasma samples for long-term storage and were then monitored for cancer occurrence over the course of 2008–2018 (Wang et al. 2009). The TZL study used PanSeer test for cancer detection. PanSeer test is a non-invasive blood test based on circulating tumour DNA methylation. It reduces the background clutter by searching for ~12,000 unique tumour-specific methylation signatures. A study by Xingdong Chen and co-workers demonstrated that using PanSeer test on 605 asymptomatic individuals, they were able to diagnose cancer markers in 191 patients up to 4 years prior to conventional diagnosis in a robust manner. These 191 people were later diagnosed with stomach, oesophageal, colorectal, lung or liver cancer within 4 years of blood draw (Chen et al. 2020). Apart from the above-mentioned protocol, FISH (fluorescence in situ hybridization), CISH (chromogenic in situ hybridization) and microarray are also

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Table 7.5 List of NAT Diagnostic test kits for the detection of cancer approved by the Center for Devices and Radiological Healtha

Cancer type Acute Myeloid Leukaemia

B-Cell Chronic Lymphocytic Leukaemia

Bladder Cancer

Breast Cancer

Product name Vysis D7S486/CEP 7 FISH Probe Kit LeukoStrat CDx FLT3 Mutation Assay Abbott RealTime IDH2 VYSIS CLL FISH PROBE KIT VYSIS CLL FISH PROBE KIT CEP 12 Spectrum Orange Direct Labelled Chromosome Enumeration DNA Probe Vysis UroVysion Bladder Cancer Recurrence Kit

Manufacturer Abbott Molecular Inc. Invivoscribe Technologies Inc. Abbott Molecular Inc. Abbott Molecular Inc.

P170005 K100015

Abbott Molecular Inc.

P150041

Vysis

K962873

Vysis

K033982, K013785, K011031 DEN170046

23andMe PGS Genetic Health Risk Report for BRCA1/BRCA2 (Selected Variants) Prosigna Breast Cancer Prognostic Gene Signature Assay MammaPrint

23andMe

INFORM HER2 Dual ISH DNA Probe Cocktail HER2 CISH pharmDxTM Kit HER2 Dual ISH DNA Probe Cocktail GeneSearch Breast Lymph Node (BLN) Test Kit Dako TOP2A FISH PharmDx Kit HER2 IQFISH PHARMDX INSITE HER-2/NEU KIT

Ventana Medical Systems, Inc. Dako Denmark A/S

SPOT-LIGHT HER2 CISH KIT INFORM HER-2/NEU

FDA Submission Number K131508 P160040

Nanostring Technologies

K130010

Agendia BV

K101454, K0 810 92, K0802 52, K070675, K062694 P100027

Ventana Medical Systems, Inc. Veridex, LLC. Dako Denmark A/S Dako Denmark A/S Biogenex Laboratories Inc. Invitrogen Corporation Vedanta Medial Systems Inc.

P100024 P190031 P060017 S001-S004 P050045 S001-S004 P040005 P040030 P050040 P940004 (continued)

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Table 7.5 (continued)

Cancer type

Chronic Myeloid Leukaemia

Colorectal Cancer

Endometrial Cancer Hematologic Malignancies

Product name DAKO HERCEPTEST PATH VYSION HER-2 DNA PROBE KIT DakoCytomation Her2 FISH pharmDx™ Kit Therascreen PIK3CA RGQ PCR Kit Quantidex qPCR BCR-ABL IS Kit Xpert BCR-ABL Ultra, GeneXpert Dx System, GeneXpert Infinity-48s and GeneXpert Infinity-80 Systems QXDx BCR-ABL %IS Kit For Use On The QXDx AutoDG DdPCR System MRDx BCR-ABL Test, MRDx BCR-ABL Test Software Cologuard Therascreen KRAS RGQ PCR Kit Therascreen BRAF V600E RGQ PCR Kit Epi ProColon® Cobas KRAS MUTATION TEST Praxis Extended RAS Panel VENTANA MMR RxDx MLL (KMT2A) BREAKAPART FISH PROBE KIT; AML1 (RUNX1) BREAKAPART FISH PROBE KIT, P53 (TP53) DELETION FISH PROBE KIT; EVI1 (MECOM) BREAKAPART FISH PROBE KIT, DEL (20Q) DELETION FISH PROBE KIT; AML1/ETO (RUNX1/RUNXIT1) TRANSLOCATION, DUAL FUSI, CBFB

Manufacturer Dako Denmark A/S Abbott Molecular Inc.

FDA Submission Number P980018 P980024

DakoCytomation Denmark A/S Qiagen GMBH

P040005

Asuragen Inc.

DEN160003

Cepheid

K190076

Bio-Rad Laboratories, Inc.

K181661

MolecularMD Corporation

K173492

Exact Sciences Corporation Qiagen Manchester Ltd.

P130017

Qiagen Manchester Ltd. Epigenomics AG Roche Molecular Systems, Inc. Illumina, Inc. Ventana Medical Systems, Inc. Cytosol Ltd.

P190001

P110027/ P110030 P190026 P130001 P140023 P160038 P200019 DEN170070

(continued)

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Table 7.5 (continued)

Cancer type

Product name (CBFB)/MYH11 TRANSLOCATION, DUAL FUSION FISH PROBE KIT,DEL(5Q) DELETION FISH PROBE KIT; DEL(7Q)DELETION FISH PROBE KIT Adaptive Biotechnologies ClonoSEQ Assay

Lung and Colon Cancer Assay Melanoma

Multiple Myeloma/Acute Lymphoblastic Leukaemia (MRD) Non-Small Cell Lung Cancer

ONCO/Reveal Dx Lung & Colon Cancer Assay Roche cobas DNA Sample Preparation Kit, COBAS 4800 BRAF V600 MUTATION TEST THXID-BRAF KIT ADAPTIVE BIOTECHNOLOGIES CLONOSEQ ASSAY

VYSIS ALK BREAK APART FISH PROBE KIT Cobas EGFR MUTATION TEST v2 THERASCREEN EGFR RGQ PCR KIT Cobas EGFR MUTATION TEST v2 Cobas EGFR MUTATION TEST v2 Oncomine Dx Target Test

Ovarian Cancer

BRACAnalysis CDx

Prostate Cancer

FoundationFocus CDxBRCA NADiA ProsVue

Prostate Cancer Predisposition

PROGENSA PCA3 Assay 23andMe PGS Genetic Risk Report For Hereditary

Manufacturer

Adaptive Biotechnologies Corporation Pillar Biosciences, Inc.

FDA Submission Number

K200009

P200011

Roche Molecular Systems, Inc.

P110020

BioMerieux, Inc. ADAPTIVE BIOTECHNOLOGIES CORPORATION

P120014 DEN170080

ABBOTT MOLECULAR, INC. Roche Molecular Systems, Inc. QIAGEN MANCHESTER LTD Roche Molecular Systems, Inc. Roche Molecular Systems, Inc. LIFE TECHNOLOGIES CORPORATION Myriad Genetic Laboratories, Inc. FOUNDATION MEDICINE, INC Iris Molecular Diagnostics Gen-Probe, Inc. 23andMe, Inc.

P110012 P120019 P120022 P150044 P150047 P160045

P140020 P160018 K101185 P100033 K211499 (continued)

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Table 7.5 (continued)

Cancer type

Product name

(HOXB13Related) Tumour Profiling

Prostate Cancer (HOXB13Related) MSK-IMPACT (Integrated Mutation Profiling Of Actionable Cancer Targets): A Hybridization-CaptureBased Next-Generation Sequencing Assay FoundationOne CDx

Memorial SloanKettering Cancer Center

DEN170058

P170019

Guardant360® CDx

Foundation Medicine, Inc. Foundation Medicine, Inc. Myriad Genetic Laboratories, Inc. NYU Langone Medical Center NantHealth, Inc. Personal Genome Diagnostics Guardant Health, Inc.

FoundationOne® Liquid CDx

Foundation Medicine, Inc.

P190032

Therascreen FGFR RGQ RT-PCR Kit

QIAGEN GmbH

P180043

FoundationOne Liquid CDx (F1 Liquid CDx) Myriad myChoice CDx NYU Langone Genome PACT Omics Core PGDx elio tissue complete Tumour Profiling; NSCLC Tumour Profiling, Liquid Biopsy Urothelial Cancer a

Manufacturer

FDA Submission Number

P200006 P190014 K202304 K190661 K192063 P200010

Data taken from US govt. FDA website (www.fda.gov)

used to detect cancer. FISH is used to detect gene rearrangements and localize the specific DNA sequences on chromosomes using fluorescence DNA probes. FISH is routinely used to detect NSCLC, haematologic malignancies and acute myeloid carcinoma (Table 7.5). CISH bypasses the requirement of fluorescence microscope and allows disclosure of chromosome translocations, chromosome number and gene amplification using standard enzymatic reactions under the brightfield microscope. Recently CISH is commercially used under brand name SPOT-LIGHT HER2 CISH KIT for detection of breast cancer by checking the changes in HER2 gene expression (Cayre et al. 2007). Microarray technology is another robust tool for cancer diagnostics as it evaluates expression levels of thousands of genes concurrently and can answer multiple diagnostic questions with one array. It is beneficial to address which genes are causing cancer and if it is due to SNP (single nucleotide polymorphism). With the data generated it becomes easier for doctors to prescribe

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gene-specific anti-cancer therapy (Liu et al. 2015). List of all the other NAT for cancer detections is given in Table 7.5.

7.3.3

Prenatal Testing

Prenatal testing comprises prenatal screening and prenatal diagnosis used for detecting complications in unborn child’s DNA. Most frequently used diagnosis techniques include amniocentesis and chorionic villus sampling (CVS). The chorionic villi are wispy projections of placental tissue that look like tiny fingers. They are formed from the fertilized egg hence contain the baby’s genetic makeup. This test can aid in the detection of some genetic illnesses, like Down syndrome or cystic fibrosis and chromosome abnormalities like aneuploidies. CVS is commonly performed at 10th–14th week of pregnancy. But various risks—including miscarriage in almost 1% population, Rh Sensitization as it might result in some of the baby’s blood cells entering mother’s circulation, bleeding, infection, rupture of membranes and other uncertain results—limit the use of CVS (Rafi and Chitty 2009). Amniocentesis is another form of invasive prenatal examination in which healthcare professionals collect small amount of amniotic fluid from uterus. It is executed during the second or third trimester of pregnancy. Amniotic fluid is the fluid surrounding the foetus which contains foetal cells and various proteins. Similar risk like CVS including amniotic fluid leakage restricts the use of amniocentesis in risk prone group of mothers. All these risks called for a non-invasive prenatal diagnosis (NIPD) and the answer to these issues was cell-free foetal DNA (cffDNA). NIPT is termed non-invasive, as it just needs collecting blood from the pregnant mother and poses no risk to the foetus. Currently cffDNA is used as a non-invasive protocol for prenatal testing. The idea was first conceptualized by Lo et al. who inspected if foetal DNA is present in maternal plasma (Lo et al. 1997). He extracted DNA from plasma, serum and nucleated blood cells from 43 pregnant women by rapid boiling method and tested the presence of male foetal DNA by Y-PCR assay in the male foetus bearing mother. Their result yielded detection of foetus-driven Y-sequences from 70% male child bearing mother. None of the control or female foetus bearing mother showed positive result. This drove the research into clinical application of using cfDNA for NIPD. cfDNA are the extracellular DNA originating from apoptosis of trophoblastic cells in placenta (Alberry et al. 2007). Another research pointed that cffDNA readily gets cleared from the maternal system once the child is delivered. This indicates that it is pregnancy specific (Chiu and Lo 2021). cffDNA screening allows one to determine chromosomal abnormalities like trisomy. It is used with very high accuracy to check for Down syndrome (trisomy 21), Edwards syndrome (trisomy 18), Patau syndrome (trisomy 13), Rh blood type and determining child’s gender (Taylor-Phillips et al. 2016; Zhang et al. 2015; Norton et al. 2015; Bianchi et al. 2014) (Table 7.6). Some research proclaims that cfDNA can also be used to detect copy number variant (CNV), though the sensitivity is too poor to use it clinically (Chitty et al. 2018). NIPT may include screening for additional chromosomal disorders that are caused by missing (deleted) or copied

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Table 7.6 Accuracy of cell-free foetal DNA-based non-invasive prenatal test (Mackie et al. 2017; Pös et al. 2019) Test FOETAL SEX RHESUS D TRISOMY 21 TRISOMY 18 TRISOMY 13 MONOSOMY X a

Sensitivity 0.989 (95% CI 0.980–0.994) 0.993 (95% CI 0.982–0.997) 0.994 (95% CI 0.983–0.998) 0.977 (95% CI 0.952–0.989) 0.906 (95% CI 0.823–0.958) 0.929 (95% CI 0.741–0.984)

Specificity 0.996 (95% CIa 0.989–0.998) 0.984 (95% CIa 0.964–0.993) 0.999 (95% CIa 0.999–1.000) 0.999 (95% CIa 0.998–1.000) 1.00 (95% CIa 0.999–0.100) 0.99 (95% CIa 0.995–0.999)

CI confidence interval

(duplicated) sections of a chromosome. As expense of genetic testing diminishes and the technology advances, researchers anticipate that NIPT will be starting to be applied for other genetic disorders testing. Several companies have started using NIPT and manufactured kits to detect aneuploidies (Table 7.7). Whole-genome next-generation sequencing (NGS) is the most preferred method as it provides a thorough interpretation of the anomalies of foetal genome compared to other methods. Several studies have shown that NGS-based NIPT can detect Down, Edwards and Patau syndromes with high accuracy and specificity (Alyafee et al. 2021; Ye et al. 2021; Jung et al. 2022). Additionally, NIPT's low-coverage (0.1) whole-genome sequencing technique presents one-of-a-kind opportunity to test for a wider range of foetal chromosomal abnormalities beyond conventional aneuploidies at a low cost. The only downside to NGS is the long time taken to show result. It requires complex algorithm and knowledge of bioinformatics to analyse the result. To address these issues recently peptide nucleic acid (PNA)-based one-step real-time polymerase chain reaction (RT-PCR) method was developed. Compared to traditional DNA probes, PNA offers faster hybridization, better stability and less sample preparation for direct detection while introducing a large variance in melting temperatures (ΔTM) between perfectly matched and single mismatched sequences (Hur et al. 2015; Kim et al. 2021; Jeong et al. 2015). They are not degraded by the DNA polymerase during PCR elongation hence form stable duplex with the target DNA fragment (Hur et al. 2015). The PNA-based RT-PCR NIPT study by Kim et al. showed 95.45% sensitivity [95% confidence interval (CI) 77.16–99.88%], 98.60% specificity (95% CI 97.66–99.23%) and 98.53% accuracy (95% CI 97.59–99.18%) for the identification of trisomy 21, 18 or 13 (Kim et al. 2021). The same was validated by another investigation by Hong et al., who confirmed that PNA-based real-time PCR assay could detect familiar trisomies with 100% sensitivity and specificity (Hong et al. 2022). The automated approach of this technique and the non-requirement of data analysis using a genetic analyser after the PCR step can provide result with much ease. This is an easy alternative to labour-intensive methods like NGS. To get rid of the complexity associated with NGS Mun Young Chang developed a protocol for NIPD using higher resolution picodroplet digital PCR (dPCR) (Chang et al. 2018; Quan et al. 2018). They applied their technique to identify autosomal recessive

Eurofins Biomnis

Atila Biosystems

Medgenome

Ninalia NIPT (Kucirka et al. 2020)

Atila Non-Invasive Prenatal Test (NIPT) Kit (Haidong et al. 2020)

Claria NIPT (Rava et al. 2014) Trisomies in chromosomes 21, 18 and 13 Rare Autosomal Aneuploidies

Trisomy 13

Trisomy 18

Disease detected Trisomy 21 and 13 Trisomy 21, 18 and 13 RAA Partial duplications and deletions ≥7 MB Trisomies 21, 18 and 13 Chromosomal imbalance greater than 7 Mb Trisomy 21 –

–

99.80%

–

>99.9%

96.4%

>99.80% >99.80%

96.4% 74.1%

95% (CI 96.3–100%) 95% (97.6–100%) 95% (97.6–100%) >99.9%

>99.90%

>99.9%

95% (CI 94.9–100%) 95% (CI 79.4–100%) 95% (73.5–100%) >99.9%

Specificity 100%

Sensitivity 100%

NGS

Highly multiplexed PCR and ddPCR

Massively-Parallel Sequencing (MPS)

Method of detection PNA-based Real-time PCR Whole-genome nextgeneration sequencing (NGS) technology

~3 h

~30 h

~26 h

Total time ~4 h

9 weeks

11 weeks

10 weeks

10 weeks

Test time (gestational age) NA

Nucleic Acid in Diagnostics

CI confidence interval, NA data not available

Company Seasun Biomaterials Illumina

Product Name Patio™ NIPT Detectiaon Kit (Jung et al. 2022) VeriSeq NIPT Solution v2 (Pertile et al. 2021)

Table 7.7 Commercially available NIPT diagnostics kits

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(AR) congenital sensorineural hearing loss. They were able to successfully predict the foetal genotype for AR monogenic illnesses caused by point mutations and establish a separate cluster for each genotype when the percentage of cffDNA was at least 6.4%. They further improved their NIPT procedure to diagnose homozygous autosomal recessive point mutations. This updated Chi-square test and Bayesian method-based protocol permitted the prenatal diagnosis using one-step picodroplet/ chip-based dPCR reactions without acquiring foetal DNA fraction (Chang et al. 2018). Mammoth innovations in the genetic diagnosis sector are leading to safer and pain free prenatal screenings by the future parents, in order to be reassured that their foetus is healthy, but the clinicians must be aware of the substantial disparity between different sequencing instruments and between different methods (NGS, dPCR, RT-PCR). It should be declared that methods used are not directly comparable as the basic principle behind the science and technology is different.

7.3.4

COVID-19

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a novel coronavirus that first made its mark in Wuhan, Hubei Province, China in December 2019, thought to be connected to a seafood market (Zhou et al. 2020). SARS-CoV2 spreads too fast from person to person and has already brought the entire world in its knees. This epidemic brings in moderate to severe symptoms in people of all age, initial sign being the pneumonia (Velavan and Meyer 2020). But apparently it causes a widespread long-lasting damage to lungs, gastrointestinal tract as well as severe disease with dyspnoea (Chan et al. 2020; Li et al. 2020; Cloutier et al. 2020). Till writing this review, the number of confirmed cases is still escalating, along with number of deaths. This number changes daily with sudden spike and sudden eclipse phase and can be tracked real time on the website hosted by Johns Hopkins University (JHU 2019). On the other hand, the high mutability rate of SARSCoV-2 makes it difficult to diagnose and difficult to target. The mutated virus even escapes the immunity (Harvey et al. 2021). Though currently there is no medicine for COVID-19 early diagnosis can prepare and with appropriate isolation can stop spreading of virus to other members of community. It is also necessary to diagnose COVID-19 infection as people from other parts of world might be carrying a mutated form of virus and if not checked it could spread to other countries. Early detection of virus is only possible by NAT. NATs, for instance RT-PCR, for SARS-CoV-2 are devised to identify viral RNA by amplifying the RNA, if any is present in a person’s sample. Augmenting those RNA allows NAT to detect very small quantities of SARS-CoV-2 RNA in a sample, making them highly sensitive for diagnosing and detection of COVID-19. Currently, NAT is the only 100% accurate method for COVID-19 detection in fast and efficient way with low false positive. Several marketed NATs that employ RT-PCR to identify the virus use many targets, so even if one of the targets is mutated, the other RT-PCR targets will still operate (Kucirka et al. 2020). High mutability rate of

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SARS-CoV-2 produces many genetic variants thus tracking multiple targets gives a high probability of identifying a genetic variants before it spreads across the globe. NAT uses variety of methods to amplify nucleic acids and detect the virus, including but not restricted to: • Reverse transcription polymerase chain reaction (RT-PCR) • Isothermal amplification including: – Nicking endonuclease amplification reaction (NEAR) – Transcription-mediated amplification (TMA) – Loop-mediated isothermal amplification (LAMP) – Helicase-dependent amplification (HDA) – Clustered regularly interspaced short palindromic repeats (CRISPR) – Strand displacement amplification (SDA) Precise and fast diagnostics for SARS-CoV-2 could benefit clinical and public health measures to handle the COVID-19 pandemic. A number of different types of NAT have been introduced since the beginning of COVID-19 pandemic and are being used in different settings such as laboratories and point-of-care (POC) platform. But it is highly essential to maintain the accuracy of these NAT. One of the biggest causes of concern for COVID-19 is its silent manifestation in a lot of asymptomatic individuals who are able to spread the virus to other individual (Almadhi et al. 2021). So, it is important to identify them in sensitive public places like airport or train stations or while crossing international borders. ID NOW by Abbott is an advanced molecular point-of-care testing technology which can return test result within 13 min. It is FDA-approved CLIA-waived instrument. ID NOW attains the quickest RNA amplification by utilizing isothermal technology, proprietary enzymes and constant temperature control. The small size also allows easy transportation of the machine. Basu et al. found good positive per cent agreement (PPA) for sample with lower CT value, however PPA fall drastically for sample with higher CT value (Basu et al. 2020). PPA of ID NOW using dry nasal swabs (54.8%) was actually poorer than another comparable RT-PCR method. Another study by Harrington et al. too decoded overall 74.7% positive agreement with the Abbott m2000 RT-PCR (Harrington et al. 2020). Based on these outcomes it can be concluded that ID NOW can be used as a rapid test for samples with high viral load, however for lower viral load the samples need to be confirmed by another utility. Xpert Xpress SARS-CoV-2 test (Cepheid, Sunnyvale, CA) is a rapid, real-time RT-PCR that is used for qualitative detection of nucleic acid in upper respiratory specimens (i.e., nasopharyngeal, oropharyngeal, nasal, or mid-turbinate swab or nasal wash/aspirate) and can generate report within 45 min. It received emergency use authorization (EUA) status on 20 March 2020. Analysis by Loeffelholz et al. decoded that the limit of detection of the Xpert test was 0.01 PFU/mL (Loeffelholz et al. 2020). Compared to all the standard-of-care methods combined, the positive agreement of the Xpert test was 99.5% (95% CI, 97.5–99.9%) and the negative agreement was 95.8% (95% CI, 92.6–97.6%). It was able to detect all replicates of

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the non-SARS Coronaviridae species and M. tuberculosis strains as SARS-CoV2 negative. Xpert Xpress SARS-CoV-2 test detects two targets, E and N2; the recognition of both targets or N2 alone is deemed positive. Hence analysis of SARS-CoV-1 generated a ‘SARS-CoV-2 presumptive positive’ test result for the reason that it sensed the E target, which is common among members of the subgenus Sarbecovirus, but not by N2. Another study demonstrated that saliva could also act as an alternative source for detecting SARS-CoV-2 RNA (McCormick-Baw et al. 2020). Jonathan J Deeks and his team analysed 78 study cohorts and reported that the average sensitivity of ID NOW was 73.0% (95% CI 66.8–78.4%) and average specificity 99.7% (95% CI 98.7–99.9%; 4 evaluations; 812 samples, 222 cases). For Xpert Xpress, the average sensitivity was 100% (95% CI 88.1–100%) and the average specificity 97.2% (95% CI 89.4–99.3%; 2 evaluations; 100 samples, 29 cases). There are several other NAT kits for SARS-CoC-2 detection (Table 7.8). Research found that Xpert Xpress had the lowest limit of detection (100% detection at 100 copies/mL), followed by ePlex (100% detection at 1000 copies/ mL) and ID NOW (20,000 copies/mL) (Zhen et al. 2020). The ePlex SARS-CoV2 Test is highly sensitive computerized qualitative nucleic acid in vitro diagnostic test that facilitates the diagnosis of COVID-19. It uses nucleic acid amplification technology using the True Sample-to-Answer Solution® ePlex instrument. This onein-all machine possesses test cartridge containing all reagents essential to extract, amplify and detect SARS-CoV-2 RNA in nasopharyngeal swab samples. The ePlex SARS-CoV-2 test revealed the assay performance of sensitivity 99.02% (94.66–99.98%; 95% CI) and specificity 98.41% (94.38–99.81%; 95% CI). It is also able to detect the newer omicron variant by analysing the N gene. The only drawback is that the reagent must be stored in 2–8 °C, which makes it difficult to place in a region without refrigeration. Too many NAT test kits are being developed globally due to its rapid demand. However, one should always keep in mind that not all give accurate result, hence proper trials must be done to ensure high error-free detection. But proper NATs with multiple targets identifying property can consistently identify small amounts of SARS-CoV-2 and are unlikely to generate a false-negative result.

7.3.5

Infectious Diseases

Nucleic acid testing has become a benchmark in clinical microbiology test sites and blood-screening centres for the detection of microbial pathogens. Despite progresses in clinical diagnosis, the clinical microbiology laboratory continues to depend extensively on traditional methods to detect micro-organisms present in clinical specimens, such as culture, microscopy and biochemical testing. It is anticipated that