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

1. Auflage 2009
ISBN: 978-0-12-380923-0
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark

E-Book, Englisch, 502 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;Conteibutors;14
6;Preface;18
7;Methods in Enzymology;20
8;Section 1: Preparative Methods for Biophysical Studies of RNA;48
8.1;Chapter 1: Large-Scale Native Preparation of In Vitro Transcribed RNA;50
8.1.1;1. Introduction;51
8.1.2;2. Native Purification of RNA: Affinity Chromatography Method;52
8.1.3;3. Native Purification of RNA: Anion-Exchange Chromatography;61
8.1.4;Acknowledgments;71
8.1.5;References;71
8.2;Chapter 2: Assembly of Complex RNAs by Splinted Ligation;74
8.2.1;1. Introduction;75
8.2.2;2. General Considerations for Splinted RNA Ligation;76
8.2.3;3. Preparation of Unmodified RNA Ligation Precursor Molecules ;78
8.2.4;4. Preparation of Modified (Dye Labeled) RNA Ligation Precursor Molecules ;84
8.2.5;5. RNA Ligation Methods;87
8.2.6;6. Application: Single-Molecule FRET Measurements;91
8.2.7;Acknowledgments;92
8.2.8;References;92
8.3;Chapter 3: Methods of Site-Specific Labeling of RNA with Fluorescent Dyes;94
8.3.1;1. Introduction;95
8.3.2;2. Design of Labeled RNA Constructs;96
8.3.3;3. Dye Labeling of RNA Fragments;100
8.3.4;4. Notes on In Vitro Transcription with T7 RNA Polymerase;102
8.3.5;5. Assembly of Labeled RNA Constructs;103
8.3.6;6.Examples of Protocols;105
8.3.7;Acknowledgments;113
8.3.8;References;113
8.4;Chapter 4: Fluorophore Labeling to Monitor tRNA Dynamics;116
8.4.1;1. Introduction;117
8.4.2;2. Methodology;122
8.4.3;3. Fluorescent Labeling of D Residues in Native and Transcripts of tRNAs;122
8.4.4;4. Fluorescent Labeling of the CCA Sequence ;129
8.4.5;5. Conclusions;136
8.4.6;Acknowledgments;137
8.4.7;References;137
8.5;Chapter 5: Use of Deoxyribozymes in RNA Research;142
8.5.1;1. Introduction;143
8.5.2;2. Deoxyribozymes for RNA Cleavage;144
8.5.3;3. Deoxyribozymes for RNA Ligation: Synthesis of Linear RNA Products;148
8.5.4;4. Deoxyribozymes for RNA Ligation: Synthesis of Branched RNA Products;154
8.5.5;5. Deoxyribozyme-Catalyzed Labeling (DECAL) of RNA;157
8.5.6;Acknowledgments;161
8.5.7;References;161
8.6;Chapter 6: Strategies in RNA Crystallography;166
8.6.1;1. Introduction;167
8.6.2;2. RNA Selection and Initial Characterization;169
8.6.3;3. Construction of Library of RNAs for Crystallization Trials;171
8.6.4;4. Improving Crystal Quality Through Postcrystal Analysis;174
8.6.5;5. Phasing Methods;177
8.6.6;6. A Case Study in the Crystallization of Lysine Riboswitch Regulatory Element;179
8.6.7;7. Concluding Remarks;181
8.6.8;Acknowledgments;182
8.6.9;References;182
9;Section 2: Biophysical Methods for RNA Conformational and Dynamics Studies;188
9.1;Chapter 7: Comparative Gel Electrophoresis Analysis of Helical Junctions in RNA;190
9.1.1;1. Principle and Theory of Electrophoresis of Bent or Kinked Nucleic Acids;191
9.1.2;2. Discrete Bending or Kinking of the Axis of a Duplex;192
9.1.3;3. The Analysis of Helical Junctions Using the Long-Short Arm Method;193
9.1.4;4. Experimental Strategies and Methods;194
9.1.5;5. Kink Turns in RNA;196
9.1.6;6. Four-Way RNA Junctions;196
9.1.7;7. Three-Way RNA Junctions;199
9.1.8;8. Complexes of Branched RNA with Proteins;202
9.1.9;Acknowledgments;202
9.1.10;References;202
9.2;Chapter 8: The Structure and Folding of Branched RNA Analyzed by Fluorescence Resonance Energy Transfer;206
9.2.1;1. Theory of FRET;209
9.2.2;2. The Possible Effects of Fluorophore Orientation;210
9.2.3;3. Choice of Fluorophores;214
9.2.4;4. Construction of Fluorophore-Labeled RNA Species;217
9.2.5;5. Steady-State Measurements of FRET;218
9.2.6;6. Time-Resolved Measurements of FRET;223
9.2.7;7. Single-Molecule FRET;226
9.2.8;Acknowledgments;229
9.2.9;References;229
9.3;Chapter 9: Analysis of RNA Folding by Native Polyacrylamide Gel Electrophoresis;236
9.3.1;1. Introduction;237
9.3.2;2. Theory of Gel Electrophoresis;238
9.3.3;3. Electrophoresis Equipment;241
9.3.4;4. Stability of Folded RNA Measured by Native PAGE;243
9.3.5;5. RNA Folding Kinetics;246
9.3.6;6. RNA Compactness and Native PAGE Mobility;247
9.3.7;7. Probing the Function of Conformers Resolved by Native PAGE;248
9.3.8;8. Controls and Further Considerations;251
9.3.9;9. Summary;252
9.3.10;Acknowledgments;252
9.3.11;References;252
9.4;Chapter 10: Using Analytical Ultracentrifugation (AUC) to Measure Global Conformational Changes Accompanying Equilibrium Tertiary Folding of RNA Molecules;256
9.4.1;1. Introduction;257
9.4.2;2. Theoretical Background;258
9.4.3;3. Materials and Instrumentation;263
9.4.4;4. Performing an RNA Folding Experiment;265
9.4.5;5. Data Analysis;271
9.4.6;6. Case Studies;277
9.4.7;7. Conclusions;279
9.4.8;Acknowledgments;280
9.4.9;References;280
9.5;Chapter 11: Use of Small Angle X-ray Scattering (SAXS) to Characterize Conformational States of Functional RNAs;284
9.5.1;1. Small Angle X-ray Scattering (SAXS) as a Tool for Global Structure Determination at Low Resolution;285
9.5.2;2. SAXS and Conformational Changes in Small Functional RNAs;285
9.5.3;3. SAXS Data Acquisition and Effects of Radiation Damage;286
9.5.4;4. SAXS Data Analysis;286
9.5.5;5. Low-Resolution Atomic Scale Models of RNA: Fitting Secondary Structure RNA Models to Three-Dimensional Shape Models;290
9.5.6;6. Determining the Thermodynamics of RNA Folding Using Bead Models;292
9.5.7;7. Concluding Remarks;295
9.5.8;Acknowledgments;296
9.5.9;References;296
9.6;Chapter 12: Time-Resolved X-ray Scattering and RNA Folding;300
9.6.1;1. Introduction ;301
9.6.2;2. SAXS Studies of RNA;301
9.6.3;3. Data Acquisition;302
9.6.4;4. Time-Resolved SAXS Methods;303
9.6.5;5. Mixer Fabrication;306
9.6.6;6. Data Analysis of SAXS Measurements;309
9.6.7;7. Concluding Remarks;313
9.6.8;Acknowledgments;313
9.6.9;References;314
9.7;Chapter 13: 2-Aminopurine as a Probe of RNA Conformational Transitions;316
9.7.1;1. Introduction;316
9.7.2;2. 2AP Structure and Photophysics;317
9.7.3;3. RNA Oligonucleotides with 2AP;320
9.7.4;4. Steady-State Fluorescence and RNA Folding;320
9.7.5;5. Time-Resolved Fluorescence Intensity Decay;325
9.7.6;Acknowledgments;331
9.7.7;References;331
9.8;Chapter 14: Fluorescence Polarization Anisotropy to Measure RNA Dynamics;334
9.8.1;1. Introduction;335
9.8.2;2. General Information of FPA Measurement;336
9.8.3;3. Choice of FPA Probes;338
9.8.4;4. Measuring FPA in a Simple Duplex, using an 11mer Control RNA Duplex as an Example;339
9.8.5;5. Use of FPA to Study Helical Dynamics of RNA, with a Junction Model Construct as an Example;341
9.8.6;6. Use of FPA to Study Helical Dynamics in a Complex RNA, with the Tetrahymena Group I Intron Ribozyme as an Example;346
9.8.7;7. Salt Dependence and Normalization of FPA with a Short Control Duplex;348
9.8.8;Acknowledgments;348
9.8.9;References;348
9.9;Chapter 15: Studying RNA Using Site-Directed Spin-Labeling and Continuous-Wave Electron Paramagnetic Resonance Spectroscopy;350
9.9.1;1. Site-Directed Spin-Labeling;351
9.9.2;2. Acquisition and Processing of cw-EPR Spectrum;355
9.9.3;3. Spectral Analysis;365
9.9.4;4. Examples of Application;367
9.9.5;Acknowledgments;373
9.9.6;References;373
9.10;Chapter 16: Mapping Global Folds of Oligonucleotides by Pulsed Electron-Electron Double Resonance;376
9.10.1;1. Introduction;377
9.10.2;2. Overview;379
9.10.3;3. The Experiment;382
9.10.4;4. Structure Generation;386
9.10.5;5. Beyond Distances;387
9.10.6;6. Comparison with Other Methods;393
9.10.7;Acknowledgments;394
9.10.8;References;394
9.11;Chapter 17: Laser-Induced Temperature Jump Infrared Measurements of RNA Folding;400
9.11.1;1. Introduction;401
9.11.2;2. Infrared Spectral Properties of RNA;403
9.11.3;3. Experimental Methods;408
9.11.4;4. Examples;413
9.11.5;5. Conclusions;418
9.11.6;References;418
10;Section 3: Electrostatics, and the Ion Atmosphere;420
10.1;Chapter 18: Probing Nucleic Acid-Ion Interactions with Buffer Exchange-Atomic Emission Spectroscopy;422
10.1.1;1. Introduction;422
10.1.2;2. Description of BE-AES;424
10.1.3;3. Buffer Equilibration;426
10.1.4;4. ICP-AES;429
10.1.5;5. Measuring Anions;430
10.1.6;6. Example Protocol;431
10.1.7;Acknowledgments;435
10.1.8;References;435
10.2;Chapter 19: Using Anomalous Small Angle X-Ray Scattering to Probe the Ion Atmosphere Around Nucleic Acids;438
10.2.1;1. Introduction;439
10.2.2;2. Background: SAXS and ASAXS;440
10.2.3;3. Experimental Setup: Samples;445
10.2.4;4. Experimental Setup: Beamline;445
10.2.5;5. Data Acquisition;447
10.2.6;6. Results and Data Analysis;450
10.2.7;7. Conclusion;454
10.2.8;Acknowledgments;456
10.2.9;References;456
10.3;Chapter 20: Simulations of RNA Interactions with Monovalent Ions;458
10.3.1;1. Introduction;459
10.3.2;2. Finite Size Artifacts in All-Atom Simulations of Ion-Nucleic Acid Interactions;461
10.3.3;3. On the Use of "Neutralizing Counterions Only" in All-Atom Simulations ;468
10.3.4;4. Results from Simulations of Canonical A-Form RNA and B-Form DNA Helices;468
10.3.5;5. Comparison with Predictions of the Nonlinear Poisson-Boltzmann Equation;473
10.3.6;Acknowledgment;477
10.3.7;References;477
10.4;Chapter 21: Ion-RNA Interactions: Thermodynamic Analysis of the Effects of Mono- and Divalent Ions on RNA Conformational Equi;480
10.4.1;1. Introduction;481
10.4.2;2. Thermodynamic Descriptions of Ion-RNA Interactions;482
10.4.3;3. Analysis of Experimental Salt Dependence Data;491
10.4.4;Acknowledgment;508
10.4.5;References;508
10.5;Chapter 22: Predicting Electrostatic Forces in RNA Folding;512
10.5.1;1. Introduction;513
10.5.2;2. Overview of Experimental Results for the Ion-Dependence of RNA Thermal Stability;514
10.5.3;3. Overview of Theoretical Methods for Predicting Ion Electrostatics for RNA Folding;518
10.5.4;4. Tightly Bound Ion Model;521
10.5.5;5. Enhancing the Computational Efficiency for the Numerical Calculations of the TBI Model ;524
10.5.6;6. Applications of the TBI Model ;525
10.5.7;7. Summary;529
10.5.8;Acknowledgments;531
10.5.9;References;531
11;Author Index;536
12;Subject Index;544
13;Color Plates;552