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

Large-Scale Native Preparation of In Vitro Transcribed RNA\r......Page 36
Introduction......Page 37
Native Purification of RNA: Affinity Chromatography Method......Page 38
Preparation of HMM protein......Page 39
Procedures......Page 40
Reagents and buffers......Page 42
Procedures......Page 43
Procedures......Page 45
Native Purification of RNA: Anion-Exchange Chromatography......Page 47
Materials......Page 48
Cell culture and plasmid purification......Page 49
In vitro RNA transcription......Page 52
Weak anion-exchange FPLC......Page 53
References......Page 57
Assembly of Complex RNAs by Splinted Ligation......Page 59
Introduction......Page 60
General Considerations for Splinted RNA Ligation......Page 61
Reagents......Page 63
Targeted RNase H cleavage of telomerase RNA......Page 65
Purification of RNase H cleavage products......Page 68
Preparation of Modified (Dye Labeled) RNA Ligation Precursor Molecules......Page 69
HPLC purification of Dye-Labeled RNA......Page 71
RNA Ligation Methods......Page 72
Reagents......Page 74
PAGE purification......Page 75
Application: Single-Molecule FRET Measurements......Page 76
References......Page 77
Methods of Site-Specific Labeling of RNA with Fluorescent Dyes......Page 79
Introduction......Page 80
Design of the construct assembly......Page 81
Selection of dyes for single molecule fluorescence studies......Page 83
Dye Labeling of RNA Fragments......Page 85
Notes on In Vitro Transcription with T7 RNA Polymerase......Page 87
Assembly of Labeled RNA Constructs......Page 88
Protocol outline......Page 90
Protocol......Page 91
Outline of the protocol......Page 93
Protocol......Page 94
Troubleshooting of ligations......Page 96
References......Page 98
Fluorophore Labeling to Monitor tRNA Dynamics......Page 101
Introduction......Page 102
Fluorescent Labeling of D Residues in Native and Transcripts of tRNAs......Page 107
Preparation of a target tRNA transcript......Page 109
The U to D conversion in the target tRNA transcript......Page 110
Fluorescent labeling of D residues......Page 112
Experimental considerations......Page 113
Fluorescent Labeling of the CCA Sequence......Page 114
Incorporation of PyC to position 75......Page 115
Experimental considerations......Page 119
Incorporation of 2AP to position 76......Page 120
Conclusions......Page 121
References......Page 122
Use of Deoxyribozymes in RNA Research......Page 126
Introduction......Page 127
Deoxyribozymes available for different RNA cleavage-site sequences......Page 128
General annealing procedure......Page 130
Analytical-scale RNA cleavage by a deoxyribozyme......Page 131
Deoxyribozymes for RNA Ligation: Synthesis of Linear RNA Products......Page 132
Deoxyribozymes available for 3\'-5\' RNA ligation\r......Page 133
Deoxyribozymes available for 2\'-5\' RNA ligation\r......Page 135
Analytical-scale RNA ligation by a deoxyribozyme......Page 136
Further efforts needed to develop deoxyribozymes for linear RNA ligation......Page 137
Deoxyribozymes for RNA Ligation: Synthesis of Branched RNA Products......Page 138
Deoxyribozymes available for 2\',5\'-branched and lariat RNA synthesis\r......Page 139
Deoxyribozyme-Catalyzed Labeling (DECAL) of RNA......Page 141
Experimental procedures for preparing the labeled tagging RNA......Page 142
Transcription of the unlabeled tagging RNA......Page 143
Experimental procedures for DECAL using the labeled tagging RNA......Page 144
References......Page 145
Strategies in RNA Crystallography......Page 149
Introduction......Page 150
Characterize the folded state of the RNA......Page 152
Consider native purification of the RNA......Page 153
Choosing phylogenetic variants of conserved function but variable periphery domains from a sequence alignment\r......Page 154
Consideration of intermolecular and intramolecular RNA packing......Page 155
Variation of peripheral helical lengths......Page 156
Cation additives to improve crystal quality......Page 157
Determine and vary the residues involved in crystal packing......Page 158
Directed mutagenesis using a crystal structure......Page 159
Phasing Methods......Page 160
Include heavy metal cations and heavy nucleotide derivatives......Page 161
A Case Study in the Crystallization of Lysine Riboswitch Regulatory Element......Page 162
Concluding Remarks......Page 164
References......Page 165
Comparative Gel Electrophoresis Analysis of Helical Junctions in RNA......Page 170
Principle and Theory of Electrophoresis of Bent or Kinked Nucleic Acids......Page 171
Discrete Bending or Kinking of the Axis of a Duplex......Page 172
The Analysis of Helical Junctions Using the Long-Short Arm Method......Page 173
Experimental Strategies and Methods......Page 174
Four-Way RNA Junctions......Page 176
Three-Way RNA Junctions......Page 179
References......Page 182
The Structure and Folding of Branched RNA Analyzed by Fluorescence Resonance Energy Transfer......Page 185
Theory of FRET......Page 188
The Possible Effects of Fluorophore Orientation......Page 189
Choice of Fluorophores......Page 193
Construction of Fluorophore-Labeled RNA Species......Page 196
Steady-State Measurements of FRET......Page 197
Time-Resolved Measurements of FRET......Page 202
Single-Molecule FRET......Page 205
References......Page 208
Analysis of RNA Folding by Native Polyacrylamide Gel Electrophoresis......Page 214
Introduction......Page 215
Mobility of macromolecules......Page 216
Chemical exchange......Page 218
Electrophoresis Equipment......Page 219
Casting and prerunning gels......Page 221
Sample preparation......Page 222
Data analysis......Page 223
RNA Folding Kinetics......Page 224
RNA Compactness and Native PAGE Mobility......Page 225
Measuring RNA activity in situ with two-dimensional PAGE......Page 226
Ligand-induced conformational change......Page 228
Controls and Further Considerations......Page 229
References......Page 230
Using Analytical Ultracentrifugation (AUC) to Measure Global Conformational Changes Accompanying Equilibrium Tertiary Folding of RNA Molecules\r......Page 234
Introduction......Page 235
Theoretical Background......Page 236
Reagents......Page 241
Analytical cell assembly......Page 242
Checking for an active self-assembly reaction......Page 243
Choosing folding conditions......Page 244
Sample preparation......Page 245
Data acquisition......Page 247
Premise of the data analysis......Page 249
Information content of the g(s) distributions......Page 250
Steps in data analysis......Page 251
tRNA......Page 255
Group I intron ribozyme......Page 256
Conclusions......Page 257
References......Page 258
Use of Small Angle X-ray Scattering (SAXS) to Characterize Conformational States of Functional RNAs......Page 262
SAXS and Conformational Changes in Small Functional RNAs......Page 263
Model free analysis......Page 264
Ab initio three-dimensional shape reconstructions......Page 266
Low-Resolution Atomic Scale Models of RNA: Fitting Secondary Structure RNA Models to Three-Dimensional Shape Models\r......Page 268
Determining the Thermodynamics of RNA Folding Using Bead Models......Page 270
Concluding Remarks......Page 273
References......Page 274
Time-Resolved X-ray Scattering and RNA Folding......Page 277
SAXS Studies of RNA......Page 278
Data Acquisition......Page 279
Stopped flow mixers and RNA folding......Page 280
Continuous flow mixers and RNA folding......Page 281
Mixer Fabrication......Page 283
X-ray beam parameters......Page 284
Samples and radiation damage......Page 285
Principal component analysis......Page 286
Three-dimensional reconstruction from one-dimensional SAXS data......Page 288
Acknowledgments......Page 290
References......Page 291
Introduction......Page 293
2AP Structure and Photophysics......Page 294
Steady-State Fluorescence and RNA Folding......Page 297
Group I ribozyme folding......Page 298
TPP riboswitch folding......Page 300
The IRE hairpin RNA loop......Page 302
Time-resolved fluorescence of the 2AP62 in the riboswitch......Page 307
References......Page 308
Fluorescence Polarization Anisotropy to Measure RNA Dynamics......Page 310
Introduction......Page 311
Choosing a wavelength for anisotropy measurement......Page 312
Factors that influence anisotropy......Page 313
Choice of FPA Probes......Page 314
Sample preparation......Page 315
FPA measurement......Page 316
Assembling the constructs without LacI......Page 317
Cell growth and induction......Page 319
FPLC purification......Page 320
Assembling model constructs with LacI and carrying out FPA measurements......Page 321
Use of FPA to Study Helical Dynamics in a Complex RNA, with the Tetrahymena Group I Intron Ribozyme as an Example\r......Page 322
References......Page 324
Studying RNA Using Site-Directed Spin-Labeling and Continuous-Wave Electron Paramagnetic Resonance Spectroscopy\r......Page 326
Site-Directed Spin-Labeling......Page 327
Spin-labeling during chemical synthesis of the nucleic acid......Page 328
Postsynthetic spin-labeling......Page 330
Acquisition and Processing of cw-EPR Spectrum......Page 331
Critical coupling of cavity......Page 333
Spectral acquisition......Page 334
Modulation phase......Page 335
Microwave power......Page 336
Scanning speed: Number of points, conversion time, and time constant......Page 337
Baseline correction......Page 338
Spectral integration......Page 339
Spectral Analysis......Page 341
Variable dynamic signatures in ligand-bound TAR RNA......Page 343
Dynamic signatures and structural requirements in the TAR-Tat interaction......Page 345
Example 2: Studying RNA/RNA interactions via cw-EPR reported τR effects\r\0......Page 347
References......Page 349
Mapping Global Folds of Oligonucleotides by Pulsed Electron-Electron Double Resonance......Page 352
Introduction......Page 353
The PELDOR pulse sequence......Page 355
The PELDOR time trace......Page 356
Extracting the distance......Page 357
Switching on the spectrometer......Page 358
Set the probe pulse frequency......Page 359
Set up the four-pulse PELDOR sequence......Page 360
The background function......Page 361
Structure Generation......Page 362
Spin counting......Page 363
Spin label orientations......Page 364
Through-bond and through-space coupling......Page 368
Comparison with Other Methods......Page 369
References......Page 370
Laser-Induced Temperature Jump Infrared Measurements of RNA Folding......Page 375
Introduction......Page 376
Sensitivity to structure......Page 378
Spectral assignments......Page 379
Thermodynamics......Page 380
Samples and sample preparation......Page 383
FTIR spectroscopy......Page 384
Time-resolved spectroscopy......Page 385
T-jump IR measurements of tRNA......Page 388
T-jump IR measurements of tetraloop formation......Page 391
References......Page 393
Introduction......Page 395
Description of BE-AES......Page 397
Buffer Equilibration......Page 399
ICP-AES......Page 402
Measuring Anions......Page 403
Example Protocol......Page 404
References......Page 408
Using Anomalous Small Angle X-Ray Scattering to Probe the Ion Atmosphere Around Nucleic Acids......Page 410
Introduction......Page 411
Anomalous SAXS provides measurements of ion distributions......Page 412
Computation and interpretation of ASAXS signal......Page 414
From source to sample......Page 417
Sample cells......Page 418
Data Acquisition......Page 419
ASAXS probes competition of different ionic species......Page 422
Comparing experimental ASAXS profiles with NLPB simulations......Page 424
Conclusion......Page 426
References......Page 428
Simulations of RNA Interactions with Monovalent Ions......Page 430
Introduction......Page 431
Finite Size Artifacts in All-Atom Simulations of Ion-Nucleic Acid Interactions......Page 433
Results from Simulations of Canonical A-Form RNA and B-Form DNA Helices......Page 440
Comparison with Predictions of the Nonlinear Poisson-Boltzmann Equation......Page 445
References......Page 449
Ion-RNA Interactions: Thermodynamic Analysis of the Effects of Mono- and Divalent Ions on RNA Conformational Equilibria\r......Page 452
Introduction......Page 453
Excess ions in an equilibrium dialysis experiment......Page 454
Thermodynamic definition of interaction coefficients......Page 458
Implications of Γ_ and Γ+ for ion activities in the presence of RNA\r\0......Page 459
Application of interaction coefficients to RNA conformational changes......Page 460
Salt dependence of RNA folding reactions......Page 463
Example 1: A-riboswitch RNA......Page 464
Example 2: tar-tar*......Page 466
Interaction coefficients in mixed divalent-monovalent cation solutions......Page 467
The linkage relation in titrations with MgCl2......Page 469
Example 1: ΔΓ2± from isothermal titration with MgCl2\r\0......Page 471
Example 2: ΔΓ2± from melting data\r\0......Page 476
Example 3: Direct measurement of Γ2±\r\0......Page 477
Comparison of three different ΔΓ2± measurement methods\r\0......Page 478
References......Page 480
Predicting Electrostatic Forces in RNA Folding......Page 483
Introduction......Page 484
Overview of Experimental Results for the Ion-Dependence of RNA Thermal Stability......Page 485
Helix-helix assembly......Page 488
Counterion condensation theory......Page 489
Size-modified Poisson-Boltzmann theory......Page 490
Correlation-corrected Poisson-Boltzmann model......Page 491
General formalism of the theory......Page 492
Free energy of the tightly bound ions......Page 493
Polarization energy of charges inside the tightly bound region......Page 494
Computation of the free energy DeltaGd for the diffusive ions......Page 495
General procedure for the numerical computations for the partition function......Page 496
Ion-binding properties......Page 498
Folding stability......Page 499
Summary......Page 500
References......Page 502