Biophysical, Chemical, and Functional Probes of RNA Structure, Interactions and Folding: Part A (eBook)

Front Cover 1
Methods in Enzymology 4
Copyright Page 5
Contents 6
Contributors 12
Preface 16
Methods in Enzymology 1
Section 1: Chemical and Functional Probing of RNA Structure, Interactions, and Folding 46
Chapter 1: Nucleotide Analog Interference Mapping 48
1. Introduction 49
2. Materials and Reagents 52
3. Methods 54
4. Properties of Analogs 64
5. Nucleotide Analog Interference Suppression 70
6. Conclusions 71
References 72
Chapter 2: Hydroxyl-Radical Footprinting to Probe Equilibrium Changes in RNA Tertiary Structure 76
1. Introduction 77
2. Sample Preparation 79
3. Equilibrium OH Footprinting Based on Peroxidative Fenton Chemistry 80
4. Equilibrium OH Footprinting Based on Oxidative Fenton Chemistry 81
5. Cleavage Product Separation 83
6. Quantitation of the Changes in the Reactivity and Data Analysis 85
7. Conclusions 89
Acknowledgments 89
References 89
Chapter 3: Rapid Quantification and Analysis of Kinetic OH Radical Footprinting Data Using SAFA 92
1. Introduction 93
2. Using SAFA 95
3. Data Normalization 102
4. Data Visualization 106
5. Conclusion 109
Acknowledgment 109
References 110
Chapter 4: High-Throughput SHAPE and Hydroxyl Radical Analysis of RNA Structure and Ribonucleoprotein Assembly 112
1. Introduction 113
2. Theory 115
3. Practice 118
4. Examples and Interpretation 123
5. Perspectives and Conclusion 131
Acknowledgments 131
References 132
Chapter 5: Metal Ion-Based RNA Cleavage as a Structural Probe 136
1. Introduction 137
2. Mechanisms of Metal Ion-Based Cleavage of Nucleic Acids 137
3. Metal Ion-Based Cleavage of RNA as a Structural Probe 140
4. Protocols 143
Acknowledgment 148
References 148
Chapter 6: 2prime-Amino-Modified Ribonucleotides as Probes for Local Interactions Within RNA 152
1. Introduction 153
2. 2prime-Amino-2prime-Deoxynucleotide Synthesis and Incorporation 155
3. 2prime-Amino-2prime-Deoxynucleotides as Sites for Covalent Modification 156
4. General Strategy for Investigating 2prime-Hydroxyl Interactions Using 2prime-Deoxy and 2prime-Aminonucleotides 157
5. Studies of RNA Catalysis Using 2prime-Amino-2prime-Deoxynucleotides 159
6. Using 2prime-Aminonucleotides to Investigate RNA Structure and Function: Case Studies 161
7. Conclusions 166
Acknowledgments 166
References 167
Chapter 7: RNA Crosslinking Methods 172
1. Introduction 173
2. Synthesis of Modified RNA Crosslinking Substrates 174
3. Generation of Crosslinked RNAs 180
4. Mapping of Crosslinked Nucleotides 184
5. Assessing the Validity of Crosslinking Data 186
References 188
Chapter 8: Chemical Probing of RNA and RNA/Protein Complexes 192
1. Introduction 193
2. Materials 195
3. Handling of the Chemicals 196
4. Optimization of the Chemical Probing Reactions 197
5. Procedure of Chemical Probing 199
6. RNA Extraction 204
7. Normalization of the RNA Sample 205
8. Primer Extension Analysis 205
9. Data Evaluation 207
10. Summary 209
Acknowledgments 209
References 209
Chapter 9: RNA Folding During Transcription: Protocols and Studies 212
1. Introduction 213
2. Protocol 1: Determination of Transcriptional Pause Sites 214
3. Protocol 2: Structural Mapping of Paused Complexes 217
4. Protocol 3: Cotranscriptional RNA Folding as Measured via Oligohybridization 219
5. Protocol 4: Cotranscriptional RNA Folding Measured via P RNA Catalytic Activity 220
6. Protocol 5: The Folding of Self-Cleaving RNAs During Transcription 224
7. Additional Methodologies 226
8. Cotranscriptional Folding Studies from Our Laboratory 226
References 235
Chapter 10: Catalytic Activity as a Probe of Native RNA Folding 240
1. Introduction 241
2. Preliminary Measurements of Catalytic Reaction 244
3. Following RNA Folding by Continuous Activity Assay 248
4. Following RNA Folding by Discontinuous Activity Assay 251
5. Other Applications of Catalytic Activity as a Probe of Folding 254
Acknowledgments 260
References 260
Chapter 11: Probing RNA Structure Within Living Cells 264
1. Introduction 265
2. Experimental Procedure 266
3. Application 279
4. Limitations 280
5. Conclusion 281
Acknowledgments 281
References 281
Chapter 12: Structural Analysis of RNA in Living Cells by In Vivo Synchrotron X-Ray Footprinting 284
1. Introduction 285
2. Beamline Setup for In Vivo Footprinting 286
3. Preparation of Samples 287
4. Exposure of Cells to X-Ray Beam 289
5. Isolation of Total RNA from Irradiated Cells 292
6. Primer Extension 293
7. Analysis of X-Ray Footprinting Experiments 296
8. Results on E. coli RNAs 298
9. Future of Footprinting 300
Acknowledgments 300
References 300
Chapter 13: Determination of Intracellular RNA Folding Rates Using Self-Cleaving RNAs 304
1. Introduction 305
2. Using RNA Turnover Rates as a "Clock" for Measuring RNA Assembly Kinetics 307
3. Applications 325
Acknowledgments 330
References 330
Section 2: Identifying Metal Ion Interactions in RNA 332
Chapter 14: Separation of RNA Phosphorothioate Oligonucleotides by HPLC 334
1. Introduction: Phosphorothioate Oligonucleotides and the Need for Separation 335
2. HPLC Separation of Phosphorothioate Diastereomers 339
3. Materials and Methods 343
4. Examples of Phosphorothioate Oligonucleotide Separations 344
Acknowledgments 352
References 352
Chapter 15: Use of Phosphorothioates to Identify Sites of Metal-Ion Binding in RNA 356
1. Introduction 357
2. Use of Phosphorothioate-Containing Ribozymes to Identify Sites of Metal-Ion Binding 357
3. Protocols 367
Acknowledgments 375
References 376
Chapter 16: EPR Methods to Study Specific Metal-Ion Binding Sites in RNA 380
1. Introduction 381
2. Room Temperature EPR Spectroscopy to Quantify Mn2+ Bound to RNA 386
3. Low-Temperature EPR Spectroscopy of Mn2+ Ions Bound to RNA 390
4. ENDOR Spectroscopy to Identify Metal Ligands 395
5. ESEEM Spectroscopy 402
6. Summary 406
Acknowledgments 409
References 409
Section 3: RNA Thermodynamics 414
Chapter 17: Optical Melting Measurements of Nucleic Acid Thermodynamics 416
1. Introduction 416
2. Instrumentation 417
3. Calibrations 418
4. Brief Theory of Optical Melting Experiments 420
5. Two-State Assumption 423
6. DeltaCpo Assumption 423
7. Experimental Design 423
8. Data Interpretation 427
9. Error Analysis 428
10. Summary 429
Acknowledgments 429
References 429
Chapter 18: Analyzing RNA and DNA Folding Using Temperature Gradient Gel Electrophoresis (TGGE) with Application to In Vitro Selections 434
1. Introduction 435
2. Temperature Gradient Gel Electrophoresis 436
3. Experimental Design and Application of TGGE to RNA and DNA 444
Acknowledgment 451
References 451
Chapter 19: Studying RNA-RNA and RNA-Protein Interactions by Isothermal Titration Calorimetry 454
1. Introduction 455
2. Required Materials 456
3. Instrumentation 456
4. Sample Considerations and Preparation 457
5. Cleaning the Sample Cell and Titration Syringe 459
6. Collecting Titration Data 460
7. Data Processing and Analysis 463
8. Special Considerations 465
9. Conclusions 466
References 467
Author Index 468
Subject Index 476
Color Plates 484