Enzymes in RNA science and biotechnology. Part A /

Detalles Bibliográficos
Clasificación:Libro Electrónico
Formato: Electrónico eBook
Idioma:Inglés
Publicado: Cambridge, MA : Academic Press, 2023.
Edición:First edition.
Colección:Methods in enzymology ; 691.
Temas:
Catalytic RNA.
Enzymes > Biotechnology.
Ribozymes.
Enzymes > Biotechnologie.
Acceso en línea:Texto completo
Tabla de Contenidos:
  • 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
  • 1 Introduction
  • 2 General methods of RNA Sanger sequencing using MarathonRT
  • 2.1 Before you begin
  • 2.2 Key resources table (Table 4)
  • 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 Polyacrylamide gel electrophoresis (PAGE) to analyze the RNA sequence
  • 2.5 Expected outcomes
  • 2.6 Advantages
  • 2.7 Limitations
  • 2.8 Optimization and troubleshooting
  • 2.8.1 Low primer extension efficiency
  • 2.8.2 Unreadable sequence
  • 3 Summary
  • Acknowledgment
  • References.
  • Chapter Three: Engineering TNA polymerases through iterative cycles of directed evolution
  • 1 Introduction
  • 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
  • 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
  • 1 Introduction
  • 2 Materials
  • 2.1 Before you begin
  • 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
  • References
  • Chapter Five: Structure-seq of tRNAs and other short RNAs in droplets and in vivo
  • 1 Introduction
  • 2 Materials
  • 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 52 triphosphates for radiolabeling
  • 3.9 Radioactive 32P 52-end labeling
  • 3.10 32-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
  • 4 Notes
  • Funding
  • References
  • Chapter Six: Capture the in vivo intact RNA structurome by CAP-STRUCTURE-seqCapture the in vivo intact RNA structurome by CAP-STRUCTURE-seq
  • 1 Introduction
  • 2 Materials
  • 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 52cap 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
  • Acknowledgments
  • Conflict of interest
  • References
  • 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
  • 1 Introduction
  • 2 Materials
  • 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
  • 4.1 Overview
  • 4.2 RNA fragmentation
  • 4.3 Traditional method
  • 4.3.1 Overview
  • 4.3.2 End-repair of RNA fragments
  • 4.3.3 52 and 32 adapter ligation
  • 4.4 Fast-forward method
  • 4.4.1 Overview
  • 4.4.2 End-repair of RNA fragments
  • 4.4.3 52 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
  • 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 templatesMaking RNA
  • 1 Introduction
  • 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
  • Acknowledgments
  • References.

Ejemplares similares