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Biophysical, chemical, and functional probes of RNA structure, interactions and folding. part A / edited by Daniel Herschlag. — Amsterdam : Elsevier, 2009. – (58.17435/C719/c.468) |
Contents
CONTENTS
Contributors
Preface
Volumes in Series
Section I. Chemical and Functional Probing of RNA structure， Interactions， and Folding
1. Nucleotide Analog Interference Mapping
1. Introduction
2. Materials and Reagents
3. Methods
4. Properties of Analogs
5. Nucleotide Analog Interference Suppression
6. Conclusions
References
2. Hydroxyl-Radical Footprinting to Probe Equilibrium Changes in RNA Tertiary Structure
1. Introduction
2. Sample Preparation
3. Equilibrium -OH Footprinting Based on Peroxidative Fenton Chemistry
4. Equilibrium -OH Footprinting Based on Oxidative Fenton Chemistry
5. Cleavage Product Separation
6. Quantitation of the Changes in the Reactivity and Data Analysis
7. Conclusions
Acknowledgments
References
3. Rapid Quantification and Analysis of Kinetic -OH Radical Footprinting Data Using SAFA
1. Introduction
2. Using SAFA
3. Data Normalization
4. Data Visualization
5. Conclusion
Acknowledgment
References
4. High-Throughput SHAPE and Hydroxyl Radical Analysis of RNA Structure and Ribonucleoprotein Assembly
1. Introduction
2. Theory
3. Practice
4. Examples and Interpretation
5. Perspectives and Conclusion
Acknowledgments
References
5. Metal Ion-Based RNA Cleavage as a Structural Probe
1. Introduction
2. Mechanisms of Metal Ion-Based Cleavage of Nucleic Acids
3. Metal Ion-Based Cleavage of RNA as a Structural Probe
4. Protocols
Acknowledgment
References
6. 2'-Amino-Modified Ribonucleotides as Probes for Local Interactions Within RNA
1. Introduction
2. 2’-Amino-2'-Deoxynucleotide Synthesis and Incorporation
3. 2’-Amino-2’-Deoxynucleotides as Sites for Covalent Modification
4. General Strategy for Investigating 2'-Hydroxyl Interactions Using 2’-Deoxy and 2’-Aminonucleotides
5. Studies of RNA Catalysis Using 2'-Amino-2’-Deoxynucleotides
6. Using 2’-Aminonucleotides to Investigate RNA Structure and Function: Case Studies
7. Conclusions
Acknowledgments
References
7. RNA Crosslinking Methods
1. Introduction
2. Synthesis of Modified RNA Crosslinking Substrates
3. Generation of Crosslinked RNAs
4. Mapping of Crosslinked Nucleotides
5. Assessing the Validity of Crosslinking Data
References
8. Chemical Probing of RNA and RNA/Protein Complexes
1. Introduction
2. Materials
3. Handling of the Chemicals
4. Optimization of the Chemical Probing Reactions
5. Procedure of Chemical Probing
6. RNA Extraction
7. Normalization of the RNA Sample
8. Primer Extension Analysis
9. Data Evaluation
10. Summary
Acknowledgments
References
9. RNA Folding During Transcription: Protocols and Studies
1. Introduction
2. Protocol 1: Determination of Transcriptional Pause Sites
3. Protocol 2: Structural Mapping of Paused Complexes
4. Protocol 3: Cotranscriptional RNA Folding as Measured via Oligohybridization
5. Protocol 4: Cotranscriptional RNA Folding Measured via P RNA Catalytic Activity
6. Protocol 5: The Folding of Self-Cleaving RNAs During Transcription
7. Additional Methodologies
8. Cotranscriptional Folding Studies from our Laboratory
References
10. Catalytic Activity as a Probe of Native RNA Folding
1. Introduction
2. Preliminary Measurements of Catalytic Reaction
3. Following RNA Folding by Continuous Activity Assay
4. Following RNA Folding by Discontinuous Activity Assay
5. Other Applications of Catalytic Activity as a Probe of Folding
Acknowledgments
References
11. Probing RNA Structure Within Living Cells
1. Introduction
2. Experimental Procedure
3. Application
4. Limitations
5. Conclusion
Acknowledgments
References
12. Structural Analysis of RNA in Living Cells by In Vivo Synchrotron X-Ray Footprinting
1. Introduction
2. Beamline Setup for In Vivo Footprinting
3. Preparation of Samples
4. Exposure of Cells to X-Ray Beam
5. Isolation of Total RNA from Irradiated Cells
6. Primer Extension
7. Analysis of X-Ray Footprinting Experiments
8. Results on E. coil RNAs
9. Future of Footprinting
Acknowledgments
References
13. Determination of Intracellular RNA Folding Rates Using Self-Cleaving RNAs
1. Introduction
2. Using RNA Turnover Rates as a "Clock" for Measuring RNA Assembly Kinetics
3. Applications
Acknowledgments
References
Section II. Identifying Metal Ion Interactions in RNA
14. Separation of RNA Phosphorothioate Oligonucleotides by HPLC
1. Introduction: Phosphorothioate Oligonucleotides and the Need for Separation
2. HPLC Separation of Phosphorothioate Diastereomers
3. Materials and Methods
4. Examples of Phosphorothioate Oligonucleotide Separations
Acknowledgments
References
15. Use of Phosphorothioates to Identify Sites of Metal-Ion Binding in RNA
1. Introduction
2. Use of Phosphorothioate-Containing Ribozymes to Identify Sites of Metal-Ion Binding
3. Protocols
Acknowledgments
References
16. EPR Methods to Study Specific Metal-Ion Binding Sites in RNA
1. Introduction
2. Room Temperature EPR Spectroscopy to Quantify Mn2+ Bound to RNA
3. Low-Temperature EPR Spectroscopy of Mn2+ Ions Bound to RNA
4. ENDOR Spectroscopy to Identify Metal Ligands
5. ESEEM Spectroscopy
6. Summary
Acknowledgments
References
Section III. RNA Thermodynamics
17. Optical Melting Measurements of Nucleic Acid Thermodynamics
1. Introduction
2. Instrumentation
3. Calibrations
4. Brief Theory of Optical Melting Experiments
5. Two-State Assumption
6. ΔC Assumption
7. Experimental Design
8. Data Interpretation
9. Error Analysis
10. Summary
Acknowledgments
References
18. Analyzing RNA and DNA Folding Using Temperature Gradient Gel Electrophoresis (TGGE) with Application to In Vitro Selections
1. Introduction
2. Temperature Gradient Gel Electrophoresis
3. Experimental Design and Application of TGGE to RNA and DNA
Acknowledgment
References
19. Studying RNA-RNA and RNA-Protein Interactions by Isothermal Titration Calorimetry
1. Introduction
2. Required Materials
3. Instrumentation
4. Sample Considerations and Preparation
5. Cleaning the Sample Cell and Titration Syringe
6. Collecting Titration Data
7. Data Processing and Analysis
8. Special Considerations
9. Conclusions
References
Author Index
Subject Index
