Enzymes in RNA Science and Biotechnology / Chun Kit Kwok and Ryota Yamagami.

Format:

Book

Author/Creator:

Kwok, Chun Kit, author.

Yamagami, Ryota, author.

Series:

Methods in Enzymology Series

Methods in Enzymology Series ; v.Volume 691

Language:

English

Subjects (All):

Catalytic RNA.

Enzymes--Biotechnology.

Enzymes.

Physical Description:

1 online resource (302 pages)

Edition:

First edition.

Place of Publication:

Cambridge, MA : Elsevier Inc., [2023]

Summary:

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.

Contents:

Front Cover

Series Page

Methods in Enzymology,

Copyright

Contents

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 RNAEnd-to-end RT-PCR of long RNA and highly structured RNA

1 Introduction

2 General methods of RT-PCR using MarathonRT

2.1 Before you begin

2.2 Key resources table (Table 2)

2.3 Materials and equipment

2.4 Step-by-step method details

2.4.1 Annealing RT primers to RNA templates

2.4.2 Preparing the reverse transcription mixture

2.4.3 Carrying out the reverse transcription

2.4.4 Programming a thermal cycler

2.4.5 Preparing the PCR mixture

2.4.6 Carrying out the PCR amplification

2.4.7 Examining the PCR products by electrophoresis

2.5 Expected outcomes

2.6 Advantages

2.7 Limitations

2.8 Optimization and troubleshooting

2.8.1 Potential problems

2.8.2 Potential solutions for optimizing the procedure

3 Summary

Acknowledgment

References

Chapter Two: RT-based Sanger sequencing of RNAs containing complex RNA repetitive elements

2 General methods of RNA Sanger sequencing using MarathonRT

2.2 Key resources table (Table 4)

2.4.4 Polyacrylamide gel electrophoresis (PAGE) to analyze the RNA sequence

2.8.1 Low primer extension efficiency

2.8.2 Unreadable sequence

References.

Chapter Three: Engineering TNA polymerases through iterative cycles of directed evolution

2 Library design

2.1 Designing a library for homologous recombination

2.2 Amplification of the expression vector for Gibson assembly

2.3 PCR cleanup and DpnI treatment

2.4 Agarose gel purification

2.5 Gibson assembly of the parent genes and linear vector and transformation

2.6 Fragment generation and homologous recombination

2.7 Gibson assembly of the recombination library and linear vector

2.8 Plasmid scale-up and purification

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

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

2 Materials

2.2 In vitro transcription and RNA purification

2.3 Gel formation, running and analysis

2.4 RTS

2.4.1 Preparation of buffer

2.4.2 Reagent and instrument

2.5 SHALiPE

2.5.1 Preparation of buffer

2.5.2 Reagent and instrument

2.5.3 RNA purification and quantification

3 Methods

3.1 In vitro transcription (timing 2 day)

3.2 Gel formation (timing 1 h)

3.3 RTS assay (timing 40 min)

3.4 SHALiPE assay (timing 1.5 h)

3.4.1 NAI reaction

3.4.2 Reverse transcription (timing 30 min)

3.5 Gel running and analysis (timing 2 h)

4 Notes

5 Summary and conclusion

Funding

Chapter Five: Structure-seq of tRNAs and other short RNAs in droplets and in vivo

2.1 General equipment

2.2 General materials

3 Methods.

3.1 Overview of tRNA Structure-seq

3.2 Preparation of reagents

3.3 PCR amplification of single-stranded DNA oligopools

3.4 BsaI restriction endonuclease treatment

3.5 In vitro transcription of RNA using hemi-duplexed DNA

3.6 In vitro transcription of RNA using BsaI-cleaved dsDNA oligopools

3.7 Purification of in vitro transcribed RNA

3.8 Removal of 5′ triphosphates for radiolabeling

3.9 Radioactive 32P 5′-end labeling

3.10 3′-end fluorescent labeling

3.11 Radiation-detected accumulation studies

3.12 Fluorescence-detected accumulation studies

3.13 Preliminary DMS reaction for tRNAs in droplets

3.14 tRNA Structure-seq in droplets (Library preparation)

3.15 tRNA Structure-seq in vivo (Library preparation)

3.16 tRNA Structure-seq analysis

Chapter Six: Capture the in vivo intact RNA structurome by CAP-STRUCTURE-seqCapture the in vivo intact RNA structurome by CAP-STRUCTURE-seq

2.1 Preparation of plant materials

2.2 RNA structure probing conditions optimization for single-hit kinetics

2.2.1 Chemical treatment

2.2.2 RNA isolation, treatment, and reverse transcription

2.2.3 Urea polyacrylamide gel electrophoresis

2.3 CAP-STRUCTURE-seq RNA preparation

2.4 CAP-STRUCTURE-seq cDNA library construction

3 Equipments

4 Methods

4.1 Overview

4.2 Plant materials and growth conditions

4.3 Gel-based 18S rRNA structure probing

4.3.1 Synthesis 2-methylnicotinic acid imidazolide (NAI)

4.3.2 Optimize the chemical treatment conditions for single-hit kinetics

4.3.3 RNA isolation

4.3.4 DNase treatment

4.3.5 First-strand cDNA synthesis with specific primer

4.3.6 Gel analysis of 18S rRNA structure and quantification

4.4 (+) SHAPE and (−) SHAPE CAP-STRUCTURE-seq RNA preparation

4.4.1 5′cap enrichment.

4.4.2 Poly (A) selection

4.5 (+) SHAPE and (−) SHAPE CAP-STRUCTURE-seq cDNA library construction

4.5.1 First strand cDNA preparation via reverse transcription

4.5.2 Single strand DNA ligation

4.5.3 cDNA size selection

4.5.4 PCR cycle optimization and PCR pool amplification

4.5.5 Size selection of PCR pool amplification products

4.6 Library validation

4.7 CAP-STRUCTURE-seq analysis

4.7.1 Deep sequencing reads mapping

4.7.2 SHAPE reactivity calculation

5 Notes

6 Concluding remarks and perspective

Conflict of interest

Chapter Seven: Sequencing-based analysis of RNA structures in living cells with 2A3 via SHAPE-MaPSequencing-based analysis of RNA structures in living cells with 2A3 via SHAPE-MaP

2.1 Equipment

2.2 Oligonucleotides and adapters

2.2.1 Barcodes

2.3 Reagents and kits

2.4 Buffers

2.4.1 Synthesis of 2-aminopyridine-3-carboxylic acid imidazolide (2A3)

2.4.2 Preparation of 2X T4 RNA Ligase Buffer

3 SHAPE probing

3.1 Overview

3.2 In-cell probing of Escherichia coli cells

3.3 RNA extraction

3.3.1 RNA quality control

4 Library preparation

4.2 RNA fragmentation

4.3 Traditional method

4.3.1 Overview

4.3.2 End-repair of RNA fragments

4.3.3 5′ and 3′ adapter ligation

4.4 Fast-forward method

4.4.1 Overview

4.4.2 End-repair of RNA fragments

4.4.3 5′ adapter ligation

4.5 Reverse transcription

4.6 Library enrichment

4.7 Library quantification and QC

5 Data analysis

5.1 Overview

5.2 Reference index preparation

5.3 Data preprocessing and read mapping

5.4 Mutation counting

5.5 Reactivity calculation

5.6 Structure prediction

5.6.1 Optimization of folding parameters

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 templatesMaking RNA

2 DNA template design for in vitro transcription

2.1 General design principle

2.2 Linearized plasmid DNA as template

2.3 PCR-amplified DNA as template

3 General methods of in vitro transcription using T7 RNA polymerase

3.1 Before you begin

3.2 Key resources table

3.3 Materials and equipment

3.4 Step-by-step method details

3.4.1 Assembling the in vitro transcription reaction mixture

3.4.2 In vitro transcription

3.4.3 Optional steps to increase in vitro transcription efficiency

3.4.4 After in vitro transcription

3.5 Expected outcomes

3.6 Advantages

3.7 Optimization and troubleshooting

3.7.1 General troubleshooting tips

3.7.2 Low transcription yield or failed transcription

3.7.3 Degradation/truncation of RNA product

3.7.4 Aggregation of RNA product

4 Preparation of specialized RNA transcripts

4.1 Preparation of radioactively labeled (body-labeled) RNA transcripts

4.1.1 Additional reagents, buffers and materials

4.1.2 Radioactive labeling reaction setup and procedure

4.1.3 Notes

4.2 Preparation of catalytic RNA molecules

4.3 Preparation of long RNA molecules

5 Purification workflow for efficient recovery of high-purity RNA transcripts

5.1 PAGE gel electrophoresis-based denaturing purification

5.1.1 Equipment

5.1.2 Reagent and buffers

5.1.3 Procedures for denaturing PAGE gel purification

5.2 Size exclusion column-based native purification

5.2.1 Equipment

5.2.2 Reagent and buffers

5.2.3 Procedures of SEC purification

6 Summary

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 DNARNA synthesis from gapped promoters.

Notes:

Includes bibliographical references.

Description based on print version record.

Description based on publisher supplied metadata and other sources.

ISBN:

0-443-15771-5

0-443-15770-7

OCLC:

1407317817