Herschlag Biophysical, Chemical, and Functional Probes of RNA Structure, Interactions and Folding: Part A

1. Auflage 2009
ISBN: 978-0-08-092213-3
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark

E-Book, Englisch, 491 Seiten

This MIE volume provides laboratory techniques that aim to predict the structure of a protein which can have tremendous implications ranging from drug design, to cellular pathways and their dynamics, to viral entry into cells.
Expert researchers introduce the most advanced technologies and techniques in protein structure and folding
Includes techniques on tiling assays.

Autoren/Hrsg.

Herschlag, Daniel

Inhaltsverzeichnis

1;Front Cover;1
2;Methods in Enzymology;4
3;Copyright Page;5
4;Contents;6
5;Contributors;12
6;Preface;16
7;Methods in Enzymology;1
8;Section 1: Chemical and Functional Probing of RNA Structure, Interactions, and Folding;46
8.1;Chapter 1: Nucleotide Analog Interference Mapping;48
8.1.1;1. Introduction;49
8.1.2;2. Materials and Reagents;52
8.1.3;3. Methods;54
8.1.4;4. Properties of Analogs;64
8.1.5;5. Nucleotide Analog Interference Suppression;70
8.1.6;6. Conclusions;71
8.1.7;References;72
8.2;Chapter 2: Hydroxyl-Radical Footprinting to Probe Equilibrium Changes in RNA Tertiary Structure;76
8.2.1;1. Introduction;77
8.2.2;2. Sample Preparation;79
8.2.3;3. Equilibrium OH Footprinting Based on Peroxidative Fenton Chemistry;80
8.2.4;4. Equilibrium OH Footprinting Based on Oxidative Fenton Chemistry;81
8.2.5;5. Cleavage Product Separation;83
8.2.6;6. Quantitation of the Changes in the Reactivity and Data Analysis;85
8.2.7;7. Conclusions;89
8.2.8;Acknowledgments;89
8.2.9;References;89
8.3;Chapter 3: Rapid Quantification and Analysis of Kinetic OH Radical Footprinting Data Using SAFA;92
8.3.1;1. Introduction;93
8.3.2;2. Using SAFA;95
8.3.3;3. Data Normalization;102
8.3.4;4. Data Visualization;106
8.3.5;5. Conclusion;109
8.3.6;Acknowledgment;109
8.3.7;References;110
8.4;Chapter 4: High-Throughput SHAPE and Hydroxyl Radical Analysis of RNA Structure and Ribonucleoprotein Assembly;112
8.4.1;1. Introduction;113
8.4.2;2. Theory;115
8.4.3;3. Practice;118
8.4.4;4. Examples and Interpretation;123
8.4.5;5. Perspectives and Conclusion;131
8.4.6;Acknowledgments;131
8.4.7;References;132
8.5;Chapter 5: Metal Ion-Based RNA Cleavage as a Structural Probe;136
8.5.1;1. Introduction;137
8.5.2;2. Mechanisms of Metal Ion-Based Cleavage of Nucleic Acids;137
8.5.3;3. Metal Ion-Based Cleavage of RNA as a Structural Probe;140
8.5.4;4. Protocols;143
8.5.5;Acknowledgment;148
8.5.6;References;148
8.6;Chapter 6: 2prime-Amino-Modified Ribonucleotides as Probes for Local Interactions Within RNA;152
8.6.1;1. Introduction;153
8.6.2;2. 2prime-Amino-2prime-Deoxynucleotide Synthesis and Incorporation;155
8.6.3;3. 2prime-Amino-2prime-Deoxynucleotides as Sites for Covalent Modification;156
8.6.4;4. General Strategy for Investigating 2prime-Hydroxyl Interactions Using 2prime-Deoxy and 2prime-Aminonucleotides;157
8.6.5;5. Studies of RNA Catalysis Using 2prime-Amino-2prime-Deoxynucleotides;159
8.6.6;6. Using 2prime-Aminonucleotides to Investigate RNA Structure and Function: Case Studies;161
8.6.7;7. Conclusions;166
8.6.8;Acknowledgments;166
8.6.9;References;167
8.7;Chapter 7: RNA Crosslinking Methods;172
8.7.1;1. Introduction;173
8.7.2;2. Synthesis of Modified RNA Crosslinking Substrates;174
8.7.3;3. Generation of Crosslinked RNAs;180
8.7.4;4. Mapping of Crosslinked Nucleotides;184
8.7.5;5. Assessing the Validity of Crosslinking Data;186
8.7.6;References;188
8.8;Chapter 8: Chemical Probing of RNA and RNA/Protein Complexes;192
8.8.1;1. Introduction;193
8.8.2;2. Materials;195
8.8.3;3. Handling of the Chemicals;196
8.8.4;4. Optimization of the Chemical Probing Reactions;197
8.8.5;5. Procedure of Chemical Probing;199
8.8.6;6. RNA Extraction;204
8.8.7;7. Normalization of the RNA Sample;205
8.8.8;8. Primer Extension Analysis;205
8.8.9;9. Data Evaluation;207
8.8.10;10. Summary;209
8.8.11;Acknowledgments;209
8.8.12;References;209
8.9;Chapter 9: RNA Folding During Transcription: Protocols and Studies;212
8.9.1;1. Introduction;213
8.9.2;2. Protocol 1: Determination of Transcriptional Pause Sites;214
8.9.3;3. Protocol 2: Structural Mapping of Paused Complexes;217
8.9.4;4. Protocol 3: Cotranscriptional RNA Folding as Measured via Oligohybridization;219
8.9.5;5. Protocol 4: Cotranscriptional RNA Folding Measured via P RNA Catalytic Activity;220
8.9.6;6. Protocol 5: The Folding of Self-Cleaving RNAs During Transcription;224
8.9.7;7. Additional Methodologies;226
8.9.8;8. Cotranscriptional Folding Studies from Our Laboratory;226
8.9.9;References;235
8.10;Chapter 10: Catalytic Activity as a Probe of Native RNA Folding;240
8.10.1;1. Introduction;241
8.10.2;2. Preliminary Measurements of Catalytic Reaction;244
8.10.3;3. Following RNA Folding by Continuous Activity Assay;248
8.10.4;4. Following RNA Folding by Discontinuous Activity Assay;251
8.10.5;5. Other Applications of Catalytic Activity as a Probe of Folding;254
8.10.6;Acknowledgments;260
8.10.7;References;260
8.11;Chapter 11: Probing RNA Structure Within Living Cells;264
8.11.1;1. Introduction;265
8.11.2;2. Experimental Procedure;266
8.11.3;3. Application;279
8.11.4;4. Limitations;280
8.11.5;5. Conclusion;281
8.11.6;Acknowledgments;281
8.11.7;References;281
8.12;Chapter 12: Structural Analysis of RNA in Living Cells by In Vivo Synchrotron X-Ray Footprinting;284
8.12.1;1. Introduction;285
8.12.2;2. Beamline Setup for In Vivo Footprinting;286
8.12.3;3. Preparation of Samples;287
8.12.4;4. Exposure of Cells to X-Ray Beam;289
8.12.5;5. Isolation of Total RNA from Irradiated Cells;292
8.12.6;6. Primer Extension;293
8.12.7;7. Analysis of X-Ray Footprinting Experiments;296
8.12.8;8. Results on E. coli RNAs;298
8.12.9;9. Future of Footprinting;300
8.12.10;Acknowledgments;300
8.12.11;References;300
8.13;Chapter 13: Determination of Intracellular RNA Folding Rates Using Self-Cleaving RNAs;304
8.13.1;1. Introduction;305
8.13.2;2. Using RNA Turnover Rates as a "Clock" for Measuring RNA Assembly Kinetics;307
8.13.3;3. Applications;325
8.13.4;Acknowledgments;330
8.13.5;References;330
9;Section 2: Identifying Metal Ion Interactions in RNA;332
9.1;Chapter 14: Separation of RNA Phosphorothioate Oligonucleotides by HPLC;334
9.1.1;1. Introduction: Phosphorothioate Oligonucleotides and the Need for Separation;335
9.1.2;2. HPLC Separation of Phosphorothioate Diastereomers;339
9.1.3;3. Materials and Methods;343
9.1.4;4. Examples of Phosphorothioate Oligonucleotide Separations;344
9.1.5;Acknowledgments;352
9.1.6;References;352
9.2;Chapter 15: Use of Phosphorothioates to Identify Sites of Metal-Ion Binding in RNA;356
9.2.1;1. Introduction;357
9.2.2;2. Use of Phosphorothioate-Containing Ribozymes to Identify Sites of Metal-Ion Binding;357
9.2.3;3. Protocols;367
9.2.4;Acknowledgments;375
9.2.5;References;376
9.3;Chapter 16: EPR Methods to Study Specific Metal-Ion Binding Sites in RNA;380
9.3.1;1. Introduction;381
9.3.2;2. Room Temperature EPR Spectroscopy to Quantify Mn2+ Bound to RNA;386
9.3.3;3. Low-Temperature EPR Spectroscopy of Mn2+ Ions Bound to RNA;390
9.3.4;4. ENDOR Spectroscopy to Identify Metal Ligands;395
9.3.5;5. ESEEM Spectroscopy;402
9.3.6;6. Summary;406
9.3.7;Acknowledgments;409
9.3.8;References;409
10;Section 3: RNA Thermodynamics;414
10.1;Chapter 17: Optical Melting Measurements of Nucleic Acid Thermodynamics;416
10.1.1;1. Introduction;416
10.1.2;2. Instrumentation;417
10.1.3;3. Calibrations;418
10.1.4;4. Brief Theory of Optical Melting Experiments;420
10.1.5;5. Two-State Assumption;423
10.1.6;6. DeltaCpo Assumption;423
10.1.7;7. Experimental Design;423
10.1.8;8. Data Interpretation;427
10.1.9;9. Error Analysis;428
10.1.10;10. Summary;429
10.1.11;Acknowledgments;429
10.1.12;References;429
10.2;Chapter 18: Analyzing RNA and DNA Folding Using Temperature Gradient Gel Electrophoresis (TGGE) with Application to In Vitro Selections;434
10.2.1;1. Introduction;435
10.2.2;2. Temperature Gradient Gel Electrophoresis;436
10.2.3;3. Experimental Design and Application of TGGE to RNA and DNA;444
10.2.4;Acknowledgment;451
10.2.5;References;451
10.3;Chapter 19: Studying RNA-RNA and RNA-Protein Interactions by Isothermal Titration Calorimetry;454
10.3.1;1. Introduction;455
10.3.2;2. Required Materials;456
10.3.3;3. Instrumentation;456
10.3.4;4. Sample Considerations and Preparation;457
10.3.5;5. Cleaning the Sample Cell and Titration Syringe;459
10.3.6;6. Collecting Titration Data;460
10.3.7;7. Data Processing and Analysis;463
10.3.8;8. Special Considerations;465
10.3.9;9. Conclusions;466
10.3.10;References;467
11;Author Index;468
12;Subject Index;476
13;Color Plates;484