Handbook of RNA Biochemistry E-Book

• Herausgeber: Wiley-VCH
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 2854

Veröffentlichungsjahr: 2015

Beschreibung

The second edition of a highly acclaimed handbook and ready reference.
Unmatched in its breadth and quality, around 100 specialists from all over the world share their up-to-date expertise and experiences, including hundreds of protocols, complete with explanations, and hitherto unpublished troubleshooting hints.
They cover all modern techniques for the handling, analysis and modification of RNAs and their complexes with proteins. Throughout, they bear the practising bench scientist in mind, providing quick and reliable access to a plethora of solutions for practical questions of RNA research, ranging from simple to highly complex.
This broad scope allows the treatment of specialized methods side by side with basic biochemical techniques, making the book a real treasure trove for every researcher experimenting with RNA.

Leseprobe

Table of Contents

Cover

Related Titles

Title Page

Copyright

Preface

List of Contributors

Part I: RNA Synthesis and Detection

Chapter 1: Enzymatic RNA Synthesis Using Bacteriophage T7 RNA Polymerase

1.1 Introduction

1.2 Description of Method – T7 Transcription

In vitro

1.3 Transcription Protocols

1.4 Troubleshooting

1.5 Rapid Preparation of T7 RNA Polymerase

References

Chapter 2: Production of RNAs with Homogeneous 5′- and 3′-Ends

2.1 Introduction

2.2 Description of Approach

2.3 Critical Experimental Steps, Changeable Parameters, Troubleshooting

2.4 PCR Protocols

2.5 Potential Problems

References

Chapter 3: RNA Ligation

3.1 General Introduction

3.2 RNA Ligation Using T4 DNA Ligase (T4 Dnl)

3.3 Simultaneous Splint Ligation of Five RNA Fragments to Generate RNAs for FRET Experiments

3.4 T4 RNA Ligase(s)

References

Chapter 4: Northern Blot Detection of Small RNAs

4.1 Introduction

4.2 Northern Hybridization Protocols

References

Chapter 5: Rapid, Non-Denaturing, Large-Scale Purification of In Vitro Transcribed RNA Using Weak Anion-Exchange Chromatography

5.1 Introduction

5.2 Materials

5.3 Protocols for Plasmid Design and Preparation, RNA Transcription, and Weak Anion-Exchange Purification

5.4 Troubleshooting

Acknowledgments

References

Chapter 6: 3′-Terminal Attachment of Fluorescent Dyes and Biotin

6.1 Introduction

6.2 Description of Method

6.3 History of the Method

6.4 Troubleshooting

Acknowledgment

References

Chapter 7: Chemical RNA Synthesis, Purification, and Analysis

7.1 Introduction

7.2 Description

7.3 Troubleshooting

References

Chapter 8: Modified RNAs as Tools in RNA Biochemistry

8.1 Introduction

8.2 Description of Methods

8.3 Experimental Protocols

References

Part II: Structure Determination

Chapter 9: Direct Determination of RNA Sequence and Modification by Radiolabeling Methods

9.1 Introduction

9.2 General Methods

9.3 Isolation of Pure RNA Species from Biological Material

9.4 Radioactive Labeling of RNA Termini

9.5 Sequencing of End-Labeled RNA

9.6 Determination of Terminal RNA Sequences by Two-dimensional Mobility Shift

9.7 Determination of Modified Nucleotides by Postlabeling Methods

9.8 Conclusions and Outlook

Acknowledgments

References

Chapter 10: Probing RNA Structure In Vitro with Enzymes and Chemicals

10.1 Introduction

10.2 Enzymatic and Chemical Probes

10.3

In Vivo

DMS Modification

10.4 Commentary

10.5 Troubleshooting

Acknowledgments

References

Chapter 11: Probing RNA Solution Structure by Photocrosslinking: Incorporation of Photoreactive Groups at RNA Termini and Determination of Crosslinked Sites by Primer Extension

11.1 Introduction

11.2 Description

11.3 Troubleshooting

11.4 Probing RNA Structure by Photoaffinity Crosslinking with 4-Thiouridine and 6-Thioguanosine

References

Chapter 12: Terbium(III) Footprinting as a Probe of RNA Structure and Metal Binding Sites

12.1 Introduction

12.2 Application Example

12.3 Troubleshooting

12.4 Frontiers in Footprinting Data Analysis

References

Chapter 13: Pb2+-Induced Cleavage of RNA

13.1 Introduction

13.2 Pb

2+

-Induced Cleavage to Probe Metal Ion Binding Sites, RNA Structure, and RNA–Ligand Interactions

13.3 Troubleshooting

Acknowledgments

References

Chapter 14: Identification and Characterization of Metal Ion Coordination Interactions with RNA by Quantitative Analysis of Thiophilic Metal Ion Rescue of Site-Specific Phosphorothioate Modifications

14.1 Introduction

14.2 Purification of Phosphorothioate Stereoisomers by RP-HPLC

14.3 Techniques for Incorporation of Phosphorothioates into RNA

14.4 Kinetic Analysis of Thiophilic Metal Ion Rescue

14.5 Data Analysis by Fitting to Simple Equilibrium Models

References

Chapter 15: Probing RNA Structure and Ligand Binding Sites on RNAby Fenton Cleavage

15.1 Introduction

15.2 Comments and Troubleshooting

References

Chapter 16: Measuring the Stoichiometry of Magnesium Ions Bound to RNA

16.1 Introduction

16.2 Separation of Free Mg

2+

from RNA-bound Mg

2+

16.3 Forced Dialysis Is the Preferred Method for Separating Bound and Free Mg

2+

16.4 Alternative Methods for Separating Free and Bound Mg

2+

Ions

16.5 Determining the Concentration of Free Mg

2+

in the Flow-Through

16.6 How to Determine the Concentration of Mg

2+

Bound to the RNA and the Number of Binding Sites on the RNA

16.7 Conclusion

16.8 Troubleshooting

References

Chapter 17: Nucleotide Analog Interference Mapping and Suppression (NAIM/NAIS): a Combinatorial Approach to Study RNA Structure, Folding, and Interaction with Proteins

17.1 Introduction

17.2 Experimental Protocols for NAIM

17.3 Experimental Protocols for NAIS

Acknowledgments

References

Chapter 18: Nucleotide Analog Interference Mapping (NAIM): Application to the RNase P System

18.1 Introduction

18.2 NAIM Analysis of

cis

-Cleaving RNase P RNA-tRNA Conjugates

18.3 Troubleshooting

References

Chapter 19: Identification of Divalent Metal Ion Binding Sites in RNA/DNA-Metabolizing Enzymes by Fe(II)-Mediated Hydroxyl Radical Cleavage

19.1 Introduction

19.2 Probing Divalent Metal Ion Binding Sites

19.3 Notes and Troubleshooting

References

Chapter 20: RNA Structure and Folding Analyzed Using Small-Angle X-Ray Scattering

20.1 Introduction

20.2 Description of Method

20.3 Troubleshooting

20.4 Conclusions – Outlook

Acknowledgments

Abbreviations

References

Chapter 21: Temperature-Gradient Gel Electrophoresis of RNA

21.1 Introduction

21.2 Method

21.3 Optimization of Experimental Conditions

21.4 TGGE – General Interpretation Rules

21.5 Examples of TGGE Applications

References

Chapter 22: UV Melting Studies with RNA

22.1 Introduction

22.2 A Simplified Account of the Physical Basis of UV Absorption

22.3 Definitions and Nomenclature

22.4 Well-Known and Less Well-Known Characteristics of UV Absorption by Nucleic Acid Bases

22.5 The Basis of UV Melting Experiments for Thermodynamic Studies

22.6 The Two-State Approximation and Its Limitations

22.7 Equilibrium and Non-equilibrium

22.8 A Common Pitfall with Self-Complementary Sequences

22.9 Extracting Thermodynamic Information from Melting Curves of Large RNAs

22.10 Parameters Influencing the Melting Temperature

22.11 Practical Problems

22.12 A Neat Experimental Solution to the Sloping Baseline

Acknowledgment

Appendix A: Difference between

T

m

and

T

max

and DMC Normalization

Appendix B: Experimental Setup against Evaporation

Appendix C: The Subtleties with Partial Derivatives for Δ

C

P

Determination

Appendix D: Buffer p

K

a

Variation with the Temperature

References

Chapter 23: RNA Crystallization

23.1 Introduction

23.2 RNA Purification

23.3 RNA Crystallization

23.4 Conclusions

References

Chapter 24: Studying RNA Using Single Molecule Fluorescence Resonance Energy Transfer

24.1 Introduction

24.2 Theory of Fluorescence Resonance Energy Transfer

24.3 Experimental Design

24.4 smFRET Experiments Using Immobilized Molecules

24.5 Troubleshooting

References

Chapter 25: Atomic Force Microscopy Imaging and Force Spectroscopyof RNA

25.1 Introduction

25.2 AFM Imaging of RNA Structures

25.3 Example Protocol: RNA Preparation for AFM Imaging in Air Using PL-Coated Mica

25.4 Troubleshooting

25.5 Force Spectroscopy AFM

25.6 Outlook

Acknowledgments

References

Part III: Structure Determination

Chapter 26: Secondary Structure Prediction

26.1 Introduction

26.2 Thermodynamics

26.3 Formal Background

26.4

mfold

and

UNAFold

26.5

RNAfold

26.6 Troubleshooting

Acknowledgment

References

Chapter 27: RNA Secondary Structure Analysis using Abstract Shapes

27.1 Introduction to Abstract Shape Analysis

27.2 Protocol 1: Computing Shape Representative Structures

27.3 Protocol 2: Probabilistic Shape Analysis

27.4 Protocol 3: Comparative Shape Analysis from Aligned Sequences

27.5 Protocol 4: Comparative Shape Analysis from Unaligned Sequences

27.6

RNAshapes

Parameter Overview

27.7

RNAlishapes

Parameter Overview

References

Chapter 28: Screening Genome Sequences for known RNA Genes or Motifs

28.1 Introduction

28.2 Choosing the Right Search Program

28.3 Overview of the RNA Search Procedure

28.4 Assessing Search Specificity

28.5 A Test Case: Looking for Homologs of a Bacterial sRNA

28.6 Conclusion

28.7 Supplemental Data

28.8 Program Versions and Download Sites

Acknowledgments

References

Chapter 29: Homology Search for Small Structured Non-Coding RNAs

29.1 Introduction

29.2 Materials

29.3 Protocol: mascRNAs

29.4 Concluding Remarks

Acknowledgments

References

Chapter 30: Predict RNA 2D and 3D Structure over the Internet using MC-Tools

30.1 Introduction

30.2 Materials

30.3 MC-Tools

30.4 Troubleshooting