Enzymes in RNA Science and Biotechnology

1st Edition, Volume 691 - October 27, 2023
Editors: Chun Kit Kwok, Ryota Yamagami
Language: English
Hardback ISBN:
9 7 8 - 0 - 4 4 3 - 1 5 7 7 0 - 7
eBook ISBN:
9 7 8 - 0 - 4 4 3 - 1 5 7 7 1 - 4

Enzymes in RNA Science and Biotechnology, Volume 691 in the Methods in Enzymology series, highlights new advances in the field, including chapters on Reverse transcriptase… Read more

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Enzymes in RNA Science and Biotechnology, Volume 691 in the Methods in Enzymology series, highlights new advances in the field, including chapters on Reverse transcriptase Part I (discovery, preparation, general utilization, MarathonRT for routine RT-PCR and for cDNA synthesis on challenging RNA templates Structured RNAs, repeat RNAs and more, Engineering TNA Polymerases Through Iterative Cycles of Directed Evolution, Reverse transcriptase Part II (RNA structure mapping and determination), RNA G-quadruplex (rG4) structure detection using RTS and SHALiPE assays, tRNA Structure-seq in vivo and in droplets, Capture the in vivo intact RNA structurome by CAP-STRUCTURE-seq, and more.

Cover image
Title page
Table of Contents
Series Page
Copyright
Contributors
Preface
Section 1 - Reverse transcriptase Part I (discovery, preparation, general utilization)
Chapter One: End-to-end RT-PCR of long RNA and highly structured RNA
Abstract
1 Introduction
2 General methods of RT-PCR using MarathonRT
3 Summary
Acknowledgment
References
Chapter Two: RT-based Sanger sequencing of RNAs containing complex RNA repetitive elements
Abstract
1 Introduction
2 General methods of RNA Sanger sequencing using MarathonRT
3 Summary
Acknowledgment
References
Chapter Three: Engineering TNA polymerases through iterative cycles of directed evolution
Abstract
1 Introduction
2 Library design
3 Cell growth, sample preparation for Droplet-based Optical Polymerase Sorting (DrOPS) and plasmid recovery
4 Colony picking and activity screen
5 Mutagenic PCR
6 Notes
7 Summary and conclusions
Acknowledgments
References
Section 2 Reverse transcriptase Part II (RNA structure mapping and determination) biosynthesis
Chapter Four: RNA G-quadruplex (rG4) structure detection using RTS and SHALiPE assays
Abstract
1 Introduction
2 Materials
3 Methods
4 Notes
5 Summary and conclusion
Funding
References
Chapter Five: Structure-seq of tRNAs and other short RNAs in droplets and in vivo
Abstract
1 Introduction
2 Materials
3 Methods
4 Notes
Funding
References
Chapter Six: Capture the in vivo intact RNA structurome by CAP-STRUCTURE-seq
Abstract
1 Introduction
2 Materials
3 Equipments
4 Methods
5 Notes
6 Concluding remarks and perspective
References
Chapter Seven: Sequencing-based analysis of RNA structures in living cells with 2A3 via SHAPE-MaP
Abstract
1 Introduction
2 Materials
3 SHAPE probing
4 Library preparation
5 Data analysis
6 Notes
7 Summary and conclusions
Funding
References
Section 3 RNA polymerase (discovery, preparation, development/engineering and application
Chapter Eight: Making RNA: Using T7 RNA polymerase to produce high yields of RNA from DNA templates
Abstract
1 Introduction
2 DNA template design for in vitro transcription
3 General methods of in vitro transcription using T7 RNA polymerase
4 Preparation of specialized RNA transcripts
5 Purification workflow for efficient recovery of high-purity RNA transcripts
6 Summary
Acknowledgments
References
Chapter Nine: A simple approach to improving RNA synthesis: Salt inhibition of RNA rebinding coupled with strengthening promoter binding by a targeted gap in the DNA
Abstract
1 Introduction
2 General methods in transcription
3 Practical considerations
4 Summary
References
Chapter Ten: Isolation of E. coli RNA polymerase transcription elongation complexes by selective solid-phase photoreversible immobilization
Abstract
1 Introduction
2 Materials and equipment
3 Methods
4 Summary and conclusions
Funding
References
Section 4 RNA ligase
Chapter Eleven: Chemical crosslinking and ligation methods for in vivo analysis of RNA structures and interactions
Abstract
Abbreviations
1 Introduction
2 Materials
3 Materials and equipment
4 Step-by-step method details
5 Expected outcomes
6 Advantages
7 Limitations
8 Optimization and troubleshooting
9 Summary
Acknowledgments
References

Chun Kit Kwok

Dr. Chun Kit Kwok obtained his B.Sc. in Chemistry (2009) from the Chinese University of Hong Kong, after completing an exchange program at University of California, Los Angeles in 2007-2008. He completed his PhD in Pennsylvania State University (2014), mentored by Professor Philip C. Bevilacqua and Professor Sarah M. Assmann. In Apr 2014, Dr. Kwok worked as a Croucher Postdoctoral Fellow in University of Cambridge under Professor Sir Shankar Balasubramanian. In Oct 2016, Dr. Kwok's joined the City University of Hong Kong as an Assistant professor and has been promoted to Associate professor in July 2021. In 2019, he was one of the recipients of the CityU President Award and Croucher Innovation Award. In 2020, Dr. Kwok's joined the State Key Laboratory of Marine Pollution, City University of Hong Kong. In 2021, Dr. Kwok was recognized as elected member of Hong Kong Young Academy of Science (YASHK) and Hong Kong Institute for Advanced Study Rising Star in Chemistry. In 2022, Dr. Kwok was awarded the NSFC Excellent Young Scientist Fund. Dr. Kwok’s current research focus is to explore the role of RNA structures and interactions in biology, especially the functions of G-quadruplex structures/interactions and non-coding RNA structures/interactions in the mammalian transcriptome and their relevance to gene regulation, RNA metabolism and diseases. Two new research directions in the Kwok lab are to develop targeting tools for detection, imaging, intervention of these important RNA structures and interactions, as well as to invent innovative nucleic acid-based technologies for sensing chemical pollutants and pathogens

Affiliations and expertise

Associate Professor, Department of Chemistry, City University of Hong Kong, Hong Kong

Ryota Yamagami

Prof. Ryota Yamagami received his Ph.D degree under the supervision of Prof. Hori at Ehime University in Japan in 2015 where he studied folate-dependent tRNA methyltransferase and tRNA modification network in Thermus thermophilus, a thermophilic bacterium. After his Ph.D course, he had worked in the lab for 1 year as a JSPS postdoctral fellow. He then joined the Bevilacqua Lab at Pennsylvania State University as a postdoctal fellow in 2016 where he has studied RNA folding.

Affiliations and expertise

Assistant Professor, Ehime University, Ehime, Japan

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