Biophysical, Chemical, and Functional Probes of RNA Structure, Interactions and Folding:, Part 1

Nucleotide Analog Interference Mapping
......Page 36
Introduction......Page 37
Chemicals......Page 40
Buffers......Page 41
Synthesis of phosphorothioate-tagged nucleoside triphosphates
......Page 42
Incorporation of phosphorothioate analogs by in vitro transcription
......Page 45
Incorporation efficiency controls......Page 48
Activity selections......Page 49
Quantitating sites of interference......Page 51
Properties of Analogs......Page 52
Ribose modifications......Page 54
Adenosine analogs......Page 56
Guanosine analogs......Page 57
Nucleotide Analog Interference Suppression
......Page 58
Conclusions......Page 59
References......Page 60
Hydroxyl-Radical Footprinting to Probe Equilibrium Changes in RNA Tertiary Structure
......Page 64
Introduction......Page 65
Sample Preparation......Page 67
Equilibrium OH Footprinting Based on Peroxidative Fenton Chemistry
......Page 68
Equilibrium bullOH Footprinting Based on Oxidative Fenton Chemistry
......Page 69
Cleavage Product Separation......Page 71
Quantitation of the Changes in the Reactivity and Data Analysis
......Page 73
References......Page 77
Rapid Quantification and Analysis of Kinetic bullOH Radical Footprinting Data Using SAFA
......Page 80
Introduction......Page 81
Using SAFA......Page 83
Lane definition and gel rectification......Page 85
Band assignment and use of a sequence file......Page 87
Single-peak fitting with SAFA......Page 88
Data normalization using invariant residues......Page 90
Generating time-progress curves and obtaining rate constants......Page 91
Data interpretation by inspection of the time-progress curves and comparison of rate constants......Page 93
Data Visualization......Page 94
Acknowledgment
......Page 97
References......Page 98
High-Throughput SHAPE and Hydroxyl Radical Analysis of RNA Structure and Ribonucleoprotein Assembly
......Page 100
Introduction......Page 101
Theory......Page 103
RNA folding......Page 106
Shape
......Page 107
Primer extension to map modification and cleavage sites
......Page 108
cDNA analysis by capillary electrophoresis
......Page 109
Normalization......Page 110
High-throughput structure analysis of protein-assisted RNA folding of the bI3 RNA
......Page 111
Identification of a misfolded free RNA state using SHAPE
......Page 113
Identification of a protein cofactor that binds the ubiquitous RNA tetraloop-receptor motif
......Page 115
Mechanism of cooperative folding of the bI3 RNP
......Page 117
Acknowledgments......Page 119
References......Page 120
Metal Ion-Based RNA Cleavage as a Structural Probe......Page 123
Mechanisms of Metal Ion-Based Cleavage of Nucleic Acids......Page 124
Probing metal ion binding sites......Page 127
Probing RNA structure and RNA-ligand interactions
......Page 128
General overview......Page 130
5prime-Radiolabeling
......Page 131
3prime-Radiolabeling
......Page 132
RNA purification......Page 133
Cleavage reactions......Page 134
References......Page 135
2prime-Amino-Modified Ribonucleotides as Probes for Local Interactions Within RNA
......Page 139
Introduction......Page 140
2prime-Amino-2prime-Deoxynucleotide Synthesis and Incorporation
......Page 142
2prime-Amino-2prime-Deoxynucleotides as Sites for Covalent Modification
......Page 143
General Strategy for Investigating 2prime-Hydroxyl Interactions Using 2prime-Deoxy and 2prime-Aminonucleotides
......Page 144
Studies of RNA Catalysis Using 2prime-Amino-2prime-Deoxynucleotides
......Page 146
Using 2prime-Aminonucleotides to Investigate RNA Structure and Function: Case Studies
......Page 148
Example: Metal coordination at the 2prime-hydroxyl of the guanosine nucleophile in the Tetrahymena group I ribozyme (Shan and Herschlag, 1999)
......Page 149
Example: Hydrogen bond donation by the cleavage site 2prime-hydroxyl in the Tetrahymena group I ribozyme (Herschlag et al., 1999)
......Page 150
Example: Investigating the electrostatic environment of the guanosine 2prime-hydroxyl in the group I ribozyme (Shan et al., 1999a)
......Page 152
Acknowledgments......Page 153
References......Page 154
RNA Crosslinking Methods......Page 158
Introduction......Page 159
Synthesis of Modified RNA Crosslinking Substrates......Page 160
Long-range photoaffinity crosslinking with azidophenacyl modifications
......Page 161
Short-range crosslinking using thionucleosides......Page 164
General considerations of reaction conditions......Page 166
Crosslinking photoactivation......Page 167
Isolation of crosslinked products......Page 169
Alkaline hydrolysis mapping......Page 170
Primer extension mapping......Page 171
Assessing the Validity of Crosslinking Data......Page 172
References......Page 174
Chemical Probing of RNA and RNA/Protein Complexes
......Page 178
Introduction......Page 179
Solutions......Page 181
Handling of the Chemicals......Page 182
Optimization of the Chemical Probing Reactions......Page 183
Protein/30S subunit complex formation
......Page 185
Modification of the complex......Page 186
Procedure of modification interference
......Page 188
Quenching of the reaction and removal of chemical reagents
......Page 189
RNA Extraction......Page 190
Primer Extension Analysis......Page 191
Data Evaluation......Page 193
References......Page 195
RNA Folding During Transcription: Protocols and Studies
......Page 197
Introduction......Page 198
Protocol 1: Determination of Transcriptional Pause Sites
......Page 199
E. coli RNA polymerase
......Page 200
B. subtilis RNA polymerase
......Page 201
Isolation of paused complexes......Page 202
Partial T1 ribonuclease cleavage......Page 203
Protocol 3: Cotranscriptional RNA Folding as Measured via Oligohybridization
......Page 204
Protocol 4: Cotranscriptional RNA Folding Measured via P RNA Catalytic Activity
......Page 205
E. coli RNA polymerase......Page 206
Renaturation of the pre-tRNA substrate......Page 207
Conditions in which [E]>[S]......Page 208
Protocol 5: The Folding of Self-Cleaving RNAs During Transcription
......Page 209
The folding of B. subtilis P RNA during transcription by T7 RNA polymerase (Pan et al., 1999)
......Page 211
The effect of transcriptional pausing on the folding of a large circularly permuted RNA (Pan et al., 1999; Wong et al., 2005)
......Page 212
The facilitation of cotranscriptional RNA folding by pausing-induced non-native structures (Wong et al., 2007)
......Page 213
The influence of pausing and small molecule cofactors on the folding of riboswitches
......Page 214
References......Page 220
Catalytic Activity as a Probe of Native RNA Folding
......Page 224
Introduction......Page 225
How to measure the catalytic reaction and establish prefolding conditions
......Page 228
Interpreting results from catalytic rate measurements
......Page 229
Following RNA Folding by Continuous Activity Assay......Page 232
How to perform experiments using the continuous assay
......Page 233
Interpreting results from the continuous assay......Page 234
Following RNA Folding by Discontinuous Activity Assay
......Page 235
How to design and perform experiments using the discontinuous assay
......Page 236
Interpreting results from the discontinuous assay
......Page 237
Assembly of RNA-protein complexes......Page 238
Native folding at equilibrium......Page 239
RNA chaperone-mediated folding and unfolding
......Page 241
References......Page 244
Probing RNA Structure Within Living Cells
......Page 248
Introduction......Page 249
Theory......Page 250
General setup......Page 251
Optimizing the individual steps of the in vivo chemical probing assay
......Page 252
DMS modification of E. coli RNA......Page 255
RNA preparation from E. coli......Page 256
Isolating RNA from yeast......Page 257
Primer extension......Page 258
Application......Page 263
Limitations......Page 264
References......Page 265
Structural Analysis of RNA in Living Cells by  In Vivo Synchrotron X-Ray Footprinting
......Page 268
Introduction......Page 269
Beamline Setup for In Vivo Footprinting......Page 270
Preparation of Samples......Page 271
Fluorescent assay for calibration of beam intensity......Page 273
Determination of optimal exposure times......Page 274
RNA isolation using the TRIzol method......Page 276
Primer Extension......Page 277
Analysis of cleaved RNA by primer extension......Page 278
Analysis of X-Ray Footprinting Experiments......Page 280
Results on E. coli RNAs......Page 282
References......Page 284
Determination of Intracellular RNA Folding Rates Using Self-Cleaving RNAs
......Page 288
Introduction......Page 289
Using RNA Turnover Rates as a "Clock" for Measuring RNA Assembly Kinetics
......Page 291
Expression of chimeric self-cleaving RNAs in yeast
......Page 294
SD medium supplemented with adenine, histidine, leucine, and tryptophan (1 L)
......Page 295
Collection of yeast samples for analysis of RNA decay rates
......Page 296
Extraction of yeast RNA and coprecipitation with hybridization probes
......Page 298
RNase protection assay......Page 300
Transcription reaction, 10 muL
......Page 302
Hybridization of radiolabeled probe to chimeric RNA
......Page 303
RNase digestion and fractionation of products
......Page 304
Ribozyme reaction buffer......Page 305
Quantification of ribozyme activity from decay time courses
......Page 306
Quantification of ribozyme activity from RNA abundance at steady state
......Page 308
Kinetic mechanism of intracellular hairpin ribozyme self-cleavage
......Page 309
Kinetics of intracellular RNA complex formation
......Page 310
Partitioning among alternative RNA secondary structures
......Page 313
References......Page 314
Separation of RNA Phosphorothioate Oligonucleotides by HPLC
......Page 316
Introduction: Phosphorothioate Oligonucleotides and the Need for Separation
......Page 317
HPLC Separation of Phosphorothioate Diastereomers
......Page 321
Elution order......Page 324
Columns......Page 325
Solvent system 1: Strong anion exchange......Page 326
Solvent system 2: Strong anion exchange......Page 329
Using complementary DNA oligonucleotides to improve RNA phosphorothioate separation
......Page 331
Summary and Conclusion......Page 332
References......Page 334
Use of Phosphorothioates to Identify Sites of Metal-Ion Binding in RNA
......Page 337
Assays based on phosphorothioate substitutions on the ribozyme
......Page 338
Thermodynamic Fingerprint Analysis (TFA) using the Tetrahymena group I ribozyme
......Page 342
Introducing phosphorothioate substitution on the ribozyme
......Page 346
General considerations on construction of phosphorothioate-containing RNA
......Page 348
Transcription of the 5prime-piece 
......Page 349
Transcription of the 3prime-piece
......Page 350
Gel purification of transcripts......Page 351
Removal of 2prime,3prime-cyclic phosphate from the 5prime-piece
......Page 352
Annealing step......Page 353
Ligation......Page 354
Kinetic assay......Page 355
Acknowledgments......Page 356
References......Page 357
EPR Methods to Study Specific Metal-Ion Binding Sites in RNA
......Page 360
Introduction......Page 361
EPR spectroscopy of Mn(II)
......Page 362
5prime-end identity
......Page 364
Mn2+ solutions......Page 365
Room Temperature EPR Spectroscopy to Quantify Mn2+ Bound to RNA
......Page 366
Sample preparation......Page 367
EPR data collection......Page 368
Data analysis......Page 369
Low-Temperature EPR Spectroscopy of Mn2+ Ions Bound to RNA
......Page 370
Sample preparation
......Page 372
EPR lineshape simulations
......Page 374
ENDOR Spectroscopy to Identify Metal Ligands
......Page 375
Deuterium substitution
......Page 377
CW ENDOR spectroscopy
......Page 378
Pulsed (ESE) ENDOR spectroscopy
......Page 379
ESEEM Spectroscopy
......Page 382
ESEEM Spectroscopy
......Page 385
Summary......Page 386
References......Page 389
Introduction......Page 393
Instrumentation......Page 394
Calibrations......Page 395
Brief Theory of Optical Melting Experiments......Page 397
Experimental Design......Page 400
Data Interpretation......Page 404
Error Analysis......Page 405
References......Page 406
Analyzing RNA and DNA Folding Using Temperature Gradient Gel Electrophoresis (TGGE) with Application to In Vitro Selections
......Page 410
Introduction......Page 411
Basic principles......Page 412
Instrumentation......Page 416
Perpendicular TGGE......Page 417
Parallel TGGE......Page 418
Preparing and running TGGE experiments......Page 419
Experimental Design and Application of TGGE to RNA and DNA......Page 420
Separation of RNA and DNA secondary structures......Page 421
TGGE-SELEX of RNA......Page 422
TGGE-SELEX of DNA......Page 425
Perpendicular TGGE melts......Page 426
References......Page 427
Studying RNA-RNA and RNA-Protein Interactions by Isothermal Titration Calorimetry
......Page 430
Introduction......Page 431
Instrumentation......Page 432
Sample Considerations and Preparation......Page 433
Stringent cleaning and nuclease contamination issues
......Page 435
Collecting Titration Data......Page 436
Data collection procedure using the single titration method
......Page 437
Data Processing and Analysis......Page 439
Temperature-dependent phenomena
......Page 441
Conclusions......Page 442
References......Page 443