Riboswitch Discovery, Structure and Function

1. Auflage 2014
ISBN: 978-0-12-801335-9
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark

E-Book, Englisch, Band 549, 570 Seiten

Reihe: Methods in Enzymology

This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers research methods in riboswitch discovery and validation, synthesis and sample prep methods for large RNAs, riboswitch structure and function methods, folding pathways and dynamics, and ligand interactions and thermodynamics. - Continues the legacy of this premier serial with quality chapters authored by leaders in the field - Covers research methods in riboswitch discovery, structure and function - Contains sections on such topics as riboswitch discovery and validation, synthesis and sample prep methods for large RNAs, riboswitch structure and function methods, folding pathways and dynamics, ligand interactions and thermodynamics

Autoren/Hrsg.

Inhaltsverzeichnis

1;Front Cover;1
2;Riboswitch Discovery, Structure and Function;4
3;Copyright;5
4;Contents;6
5;Contributors;14
6;Preface;20
6.1;Volume 1;20
6.2;Volume 2;22
7;Part I: Riboswitch Discovery;26
7.1;Chapter One: Riboswitch Discovery by Combining RNA-Seq and Genome-Wide Identification of Transcriptional Start Sites;28
7.1.1;1. Introduction;29
7.1.2;2. RNA Isolation and mRNA Enrichment;32
7.1.2.1;2.1. Equipment and materials;32
7.1.2.2;2.2. RNA isolation and quality control;37
7.1.2.3;2.3. mRNA enrichment and quality control;38
7.1.3;3. Genome-Wide Mapping of Transcription Start Sites by dRNA-Seq;39
7.1.3.1;3.1. Equipment and materials;39
7.1.3.2;3.2. Hydrolysis of triphosphate groups at mRNA 5-ends by TAP;40
7.1.3.3;3.3. Ligation of adapter on 5-end of mRNAs;40
7.1.3.4;3.4. cDNA first strand synthesis by random priming;41
7.1.3.5;3.5. cDNA sizing on agarose gels;41
7.1.3.6;3.6. PCR amplification;42
7.1.3.7;3.7. Purification of the PCR products on Agencourt AMPure beads;43
7.1.3.8;3.8. Quality control of the libraries;44
7.1.3.9;3.9. Data analysis;44
7.1.4;4. Genome-Wide Analysis of Transcript Length by RNA-Seq;45
7.1.4.1;4.1. Strand-specific RNA-Seq library construction;45
7.1.4.2;4.2. Nonoriented whole-transcript RNA-Seq library preparation;46
7.1.5;5. Processing and Analysis of dRNA-Seq and RNA-Seq Data;47
7.1.5.1;5.1. Softwares and supplementary files required for the analysis;47
7.1.5.2;5.2. Protocol;48
7.1.5.2.1;5.2.1. Trimming of the adapter sequences;48
7.1.5.2.2;5.2.2. Creation of a reference index for bowtie 1;48
7.1.5.2.3;5.2.3. Alignment of the reads to the reference genome;49
7.1.5.2.4;5.2.4. Conversion of the SAM file to a sorted BAM file and creation of an index;49
7.1.5.2.5;5.2.5. Visualization of the alignments on a genome browser;49
7.1.6;6. Characterization of New Potential Riboswitches Using dRNA-Seq and RNA-Seq Analyses;49
7.1.7;References;51
7.2;Chapter Two: Discovering Human RNA Aptamers by Structure-Based Bioinformatics and Genome-Based In Vitro Selection;54
7.2.1;1. Introduction;54
7.2.2;2. Precautions;57
7.2.3;3. Generating a Human Genomic DNA Pool;57
7.2.3.1;3.1. Materials;57
7.2.3.1.1;3.1.1. High molecular weight human genomic DNA;57
7.2.3.1.2;3.1.2. Adapter oligonucleotide sequences;57
7.2.3.1.3;3.1.3. Enzymes;58
7.2.3.1.4;3.1.4. Buffers;59
7.2.3.1.4.1;3.1.4.1. Tris/borate/EDTA buffer (10x);59
7.2.3.1.5;3.1.5. Instruments and miscellaneous;59
7.2.3.2;3.2. Procedures;59
7.2.3.2.1;3.2.1. Preparation of genomic DNA;59
7.2.3.2.2;3.2.2. Repairing genomic DNA ends;59
7.2.3.2.3;3.2.3. Addition of 5 phosphate group onto genomic DNA;60
7.2.3.2.4;3.2.4. Addition of 3 dA overhangs;60
7.2.3.2.5;3.2.5. Adapter ligation;60
7.2.3.2.6;3.2.6. PCR amplification;61
7.2.4;4. In Vitro Selection of RNA Aptamers;61
7.2.4.1;4.1. Materials;61
7.2.4.1.1;4.1.1. Selection buffers;61
7.2.4.1.2;4.1.2. Polyacrylamide gel electrophoresis;62
7.2.4.1.3;4.1.3. Agarose gel electrophoresis;62
7.2.4.1.4;4.1.4. Transcription;62
7.2.4.1.5;4.1.5. Reverse transcription;63
7.2.4.1.6;4.1.6. Polymerase chain reaction;63
7.2.4.1.7;4.1.7. Enzymes;63
7.2.4.1.8;4.1.8. Affinity column for in vitro selection;63
7.2.4.2;4.2. Procedure;63
7.2.4.2.1;4.2.1. Transcription;63
7.2.4.2.2;4.2.2. Purification of transcribed product;64
7.2.4.2.3;4.2.3. In vitro selection of RNA aptamers;66
7.2.4.2.4;4.2.4. Reverse transcription of selected RNAs;67
7.2.4.2.5;4.2.5. Polymerase chain reaction;67
7.2.5;5. Structure-Based Searches for Naturally Occurring Aptamers;69
7.2.5.1;5.1. Materials;69
7.2.5.1.1;5.1.1. Unix compliant operating system;69
7.2.5.1.2;5.1.2. RNABOB;69
7.2.5.1.3;5.1.3. RNArobo;69
7.2.5.2;5.2. Procedures;69
7.2.5.2.1;5.2.1. Descriptor;69
7.2.5.2.2;5.2.2. Sequence data;70
7.2.6;References;70
8;Part II: Synthesis and Sample Prep Methods for Large RNAs;72
8.1;Chapter Three: Affinity Purification of In Vitro Transcribed RNA with Homogeneous Ends Using a 3-ARiBo Tag;74
8.1.1;1. Introduction;75
8.1.2;2. Batch Affinity Purification of RNA Using a 3-ARiBo Tag;77
8.1.2.1;2.1. General scheme;77
8.1.2.2;2.2. Designing the ARiBo-fusion RNA;78
8.1.2.3;2.3. Cloning of the plasmid DNA template;79
8.1.2.4;2.4. Bacterial cell culture and plasmid preparation;80
8.1.2.5;2.5. In vitro transcription of RNA and optimization of glmS cleavage conditions;81
8.1.2.6;2.6. Batch affinity purification;82
8.1.2.7;2.7. Quantitative analyses for batch affinity purification using a 3-ARiBo tag;85
8.1.2.7.1;2.7.1. Denaturing gel electrophoresis;86
8.1.2.7.2;2.7.2. Quantitative analysis of ARiBo-fusion RNA produced by in vitro transcription;87
8.1.2.7.3;2.7.3. Quantitative analysis of glmS cleavage in the transcription reaction;87
8.1.2.7.4;2.7.4. Quantitative analysis of batch affinity purification;88
8.1.2.8;2.8. Troubleshooting;89
8.1.3;3. Ensuring 5-Homogeneity of Affinity-Purified RNA;92
8.1.3.1;3.1. General considerations in the selection of 5-sequences;94
8.1.3.2;3.2. Affinity purification of RNA using a 5-CRISPR tag and a 3-ARiBo tag;96
8.1.3.2.1;3.2.1. Bacterial expression of the Cse3 endonuclease;97
8.1.3.2.2;3.2.2. Purification of the Cse3 endonuclease;97
8.1.3.2.3;3.2.3. Cse3 endonuclease cleavage of the CRISPR-RNA-ARiBo precursor;100
8.1.3.3;3.3. Affinity purification of RNA using a 5-HH and a 3-ARiBo tag;101
8.1.3.4;3.4. Quantitative analyses when using a 5-tag;104
8.1.3.4.1;3.4.1. Quantitative analysis of 5-tag cleavage in the transcription reaction;104
8.1.3.4.2;3.4.2. Quantitative analysis of batch affinity purification;104
8.1.4;4. Summary;105
8.1.5;Acknowledgments;106
8.1.6;References;106
8.2;Chapter Four: Deoxyribozyme-Mediated Ligation for Incorporating EPR Spin Labels and Reporter Groups into RNA;110
8.2.1;1. Introduction;111
8.2.2;2. Synthesis of Spin-Labeled RNA Using Convertible Nucleosides;112
8.2.3;3. DNA-Catalyzed Ligation of RNA Using 9DB1*;115
8.2.3.1;3.1. General protocol for DNA-catalyzed RNA ligation on analytical scale for testing ligation sites and screening of liga...;117
8.2.3.1.1;3.1.1. Reagents;117
8.2.3.1.2;3.1.2. Procedure;118
8.2.4;4. Protocols for Synthesis of Spin-Labeled SAM-I Riboswitch;118
8.2.4.1;4.1. Synthesis of spin-labeled RNA (acceptor substrate);119
8.2.4.1.1;4.1.1. Reagents;120
8.2.4.1.2;4.1.2. Procedure;120
8.2.4.2;4.2. In vitro transcription of donor substrate;121
8.2.4.2.1;4.2.1. Reagents;121
8.2.4.2.2;4.2.2. Procedure;122
8.2.4.3;4.3. Preparative DNA-catalyzed ligation of SAM-I RNA fragments;122
8.2.4.3.1;4.3.1. Reagents;123
8.2.4.3.2;4.3.2. Procedure;123
8.2.5;5. General Considerations and Future Developments;124
8.2.5.1;5.1. Choice of label position;124
8.2.5.2;5.2. Number and type of labels;124
8.2.5.3;5.3. Position and sequence context of ligation junction;125
8.2.5.4;5.4. Alternative DNA-catalyzed approaches for site-specific labeling of RNA;126
8.2.6;Acknowledgments;126
8.2.7;References;126
8.3;Chapter Five: A Flexible, Scalable Method for Preparation of Homogeneous Aminoacylated tRNAs;130
8.3.1;1. Introduction;130
8.3.2;2. Methods;132
8.3.2.1;2.1. tRNA aminoacylation using the flexizyme;132
8.3.2.2;2.2. Chemical protection of the aminoacyl bond;134
8.3.2.3;2.3. Purification of protected aminoacylated tRNA and deprotection;136
8.3.3;Acknowledgments;137
8.3.4;References;137
8.4;Chapter Six: Synthesis of a Biotinylated Photocleavable Nucleotide Monophosphate for the Preparation of Natively Folded RNAs;140
8.4.1;1. Theory;141
8.4.2;2. Equipment;143
8.4.3;3. Materials;143
8.4.3.1;3.1. Stock solutions and buffers;145
8.4.4;4. Protocol;146
8.4.4.1;4.1. Duration;146
8.4.4.2;4.2. Preparation;147
8.4.4.3;4.3. Caution;147
8.4.5;5. Step 1: Synthesis of Biotin-PC GMP;148
8.4.5.1;5.1. Overview;148
8.4.5.2;5.2. Duration;148
8.4.5.2.1;5.2.1. Synthesis of PC alkyne;148
8.4.5.2.1.1;5.2.1.1. Tip;149
8.4.5.2.1.2;5.2.1.2. Tip;149
8.4.5.2.1.3;5.2.1.3. Tip;149
8.4.5.2.2;5.2.2. Synthesis of PC alkyne GMP;149
8.4.5.2.2.1;5.2.2.1. Tip;150
8.4.5.2.3;5.2.3. Synthesis of biotin-PC GMP;150
8.4.5.2.3.1;5.2.3.1. Tip;151
8.4.5.2.3.2;5.2.3.2. Tip;151
8.4.6;6. Step 2: Transcription of D5 and Ribosomal A-site RNAs Using Unmodified GTP and Biotin-PC GMP;151
8.4.6.1;6.1. Overview;151
8.4.6.2;6.2. Duration and transcription optimization;151
8.4.6.3;6.3. Tip;153
8.4.6.4;6.4. Tip;153
8.4.7;7. Step 3: Purification of Biotin-Labeled RNA with Affinity Avidin Column and Photocleavage;153
8.4.7.1;7.1. Overview;153
8.4.7.2;7.2. Duration;154
8.4.7.3;7.3. Tip;154
8.4.7.4;7.4. Tip;154
8.4.8;8. Conclusions;154
8.4.9;Acknowledgments;156
8.4.10;References;156
8.5;Chapter Seven: Chemo-Enzymatic Synthesis of Selectively 13C/15N-Labeled RNA for NMR Structural and Dynamics Studies;158
8.5.1;1. Theory;160
8.5.2;2. Equipment;163
8.5.3;3. Materials;164
8.5.3.1;3.1. Solutions and buffers;166
8.5.4;4. Protocol;169
8.5.5;5. Step 1: Synthesis of Uracil;170
8.5.5.1;5.1. Overview;170
8.5.5.2;5.2. Duration;170
8.5.5.3;5.3. Tip;172
8.5.5.4;5.4. Tip;172
8.5.5.5;5.5. Tip;172
8.5.5.6;5.6. Tip;172
8.5.6;6. Step 2: Synthesis of UTP;173
8.5.6.1;6.1. Overview;173
8.5.6.2;6.2. Duration;173
8.5.6.3;6.3. Tip;174
8.5.6.4;6.4. Tip;174
8.5.6.5;6.5. Tip;174
8.5.6.6;6.6. Tip;174
8.5.6.7;6.7. Tip;175
8.5.7;7. Step 3: Synthesis of CTP;175
8.5.7.1;7.1. Overview;175
8.5.7.2;7.2. Duration;175
8.5.7.3;7.3. Tip;176
8.5.7.4;7.4. Tip;176
8.5.7.5;7.5. Tip;176
8.5.8;8. Step 4: Purification and Quantification;176
8.5.8.1;8.1. Overview;176
8.5.8.2;8.2. Duration;176
8.5.8.3;8.3. Tip;178
8.5.8.4;8.4. Tip;178
8.5.8.5;8.5. Tip;178
8.5.8.6;8.6. Tip;178
8.5.8.7;8.7. Tip;178
8.5.8.8;8.8. Tip;178
8.5.9;9. Step 5: Quality Control;179
8.5.9.1;9.1. Overview;179
8.5.9.2;9.2. Duration;179
8.5.9.3;9.3. Tip;180
8.5.9.4;9.4. Tip;180
8.5.10;10. Step 6: In Vitro RNA Transcription;180
8.5.10.1;10.1. Overview;180
8.5.10.2;10.2. Duration;181
8.5.10.3;10.3. Tip;182
8.5.10.4;10.4. Tip;182
8.5.11;11. Step 7: NMR Applications;182
8.5.11.1;11.1. Overview;182
8.5.11.2;11.2. Heteronuclear single quantum coherence (HSQC);183
8.5.11.3;11.3. Transverse relaxation optimized spectroscopy (TROSY);183
8.5.12;12. Conclusion;183
8.5.13;Acknowledgments;186
8.5.14;References;186
9;Part III: Structure and Folding;188
9.1;Chapter Eight: SHAPE Analysis of Small RNAs and Riboswitches;190
9.1.1;1. Theory;191
9.1.2;2. Equipment;192
9.1.3;3. Materials;192
9.1.3.1;3.1. Solutions and buffers;193
9.1.4;4. Protocol;194
9.1.4.1;4.1. Preparation;194
9.1.4.2;4.2. Duration;195
9.1.5;5. Step 1: RNA Folding and SHAPE Probing;195
9.1.5.1;5.1. Overview;195
9.1.5.2;5.2. Duration;196
9.1.5.3;5.3. Tip;197
9.1.5.4;5.4. Tip;197
9.1.5.5;5.5. Tip;197
9.1.6;6. Step 2: Primer Extension;197
9.1.6.1;6.1. Overview;197
9.1.6.2;6.2. Duration;197
9.1.6.3;6.3. Tip;198
9.1.6.4;6.4. Tip;198
9.1.6.5;6.5. Tip;199
9.1.6.6;6.6. Tip;199
9.1.7;7. Step 3: Capillary Electrophoresis;199
9.1.7.1;7.1. Overview;199
9.1.7.2;7.2. Duration;199
9.1.7.3;7.3. Tip;199
9.1.7.4;7.4. Tip;200
9.1.7.5;7.5. Tip;200
9.1.8;8. Step 4: Data Processing Using QuShape;200
9.1.8.1;8.1. Overview;200
9.1.8.2;8.2. Duration;200
9.1.8.3;8.3. Tip;205
9.1.8.4;8.4. Tip;205
9.1.8.5;8.5. Tip;206
9.1.8.6;8.6. Tip;206
9.1.8.7;8.7. Tip;206
9.1.8.8;8.8. Tip;206
9.1.8.9;8.9. Tip;207
9.1.9;9. Step 5: Data Processing and RNA Modeling;207
9.1.9.1;9.1. Overview;207
9.1.9.2;9.2. Duration;207
9.1.9.3;9.3. Tip;209
9.1.9.4;9.4. Tip;211
9.1.9.5;9.5. Tip;211
9.1.10;Acknowledgments;211
9.1.11;References;211
9.2;Chapter Nine: Experimental Approaches for Measuring pKas in RNA and DNA;214
9.2.1;1. Introduction;215
9.2.2;2. Experimental Parameters for pH Titrations;218
9.2.2.1;2.1. Potential pitfalls: pH-promoted RNA unfolding, RNA degradation, and poor baselines;218
9.2.2.2;2.2. Choosing the pH probe and meter;221
9.2.2.3;2.3. Whether to use a buffer;222
9.2.2.4;2.4. Corrections to the pH meter reading and the use of pH paper;223
9.2.2.5;2.5. Choosing an experimental method and assigning the pKa;223
9.2.3;3. RNA Cleavage Kinetics;224
9.2.3.1;3.1. Ribozyme cleavage;224
9.2.3.2;3.2. Chimeric oligonucleotide cleavage;228
9.2.4;4. Spectroscopic-Detected Methods;229
9.2.4.1;4.1. General considerations for spectroscopic-detected pH titrations;229
9.2.4.2;4.2. UV absorbance-detected pH titrations;230
9.2.4.3;4.3. Fluorescence-detected pH titrations;232
9.2.4.4;4.4. NMR-detected pH titrations;235
9.2.4.5;4.5. Raman crystallography pH titrations;239
9.2.5;5. Perspective;240
9.2.6;Acknowledgments;241
9.2.7;References;241
9.3;Chapter Ten: Crystallographic Analysis of TPP Riboswitch Binding by Small-Molecule Ligands Discovered Through Fragment-Ba...;246
9.3.1;1. Introduction;247
9.3.2;2. Methods;248
9.3.2.1;2.1. Growth of riboswitch-fragment co-crystals;248
9.3.2.1.1;2.1.1. Considerations in transcription template and RNA construct design;249
9.3.2.1.2;2.1.2. Considerations in fragment selection;249
9.3.2.1.3;2.1.3. In vitro transcription of TPP riboswitch RNA;251
9.3.2.1.4;2.1.4. Initial screens of crystallization conditions with the cognate ligand;251
9.3.2.1.5;2.1.5. Initial screens for fragment co-crystals;252
9.3.2.1.6;2.1.6. Development of cryoprotectant solutions for vitrification of fragment co-crystals;252
9.3.2.2;2.2. Structure solution by molecular replacement;254
9.3.2.2.1;2.2.1. Structure solution by molecular replacement;255
9.3.2.2.2;2.2.2. Model building and refinement;255
9.3.2.2.3;2.2.3. Building the fragment into the model;255
9.3.3;3. Conclusions;256
9.3.4;Acknowledgments;256
9.3.5;References;256
9.4;Chapter Eleven: Methods for Using New Conceptual Tools and Parameters to Assess RNA Structure by Small-Angle X-Ray Scattering;260
9.4.1;1. Introduction;261
9.4.2;2. Specialized Equipment;263
9.4.3;3. Preparation of the RNA for a SAXS Study;263
9.4.3.1;3.1. Assessing the folded state of the RNA;263
9.4.3.2;3.2. Importance of buffer subtraction;269
9.4.4;4. Interpretation of the X-Ray Scattering Curve;270
9.4.4.1;4.1. Quantitating compactness;271
9.4.4.2;4.2. SAXS invariants;274
9.4.4.3;4.3. Real-space parameters;274
9.4.4.4;4.4. Dimensionless Kratky plot;275
9.4.5;5. Case Studies;277
9.4.5.1;5.1. SAM-I riboswitch;278
9.4.5.2;5.2. LYS riboswitch;279
9.4.6;6. Multiphase Volumetric Modeling;280
9.4.6.1;6.1. B12 riboswitch;281
9.4.7;7. Gold Labels and Comprehensive Conformations;281
9.4.8;8. Considerations;283
9.4.9;Acknowledgments;285
9.4.10;References;285
10;Part IV: Dynamics;290
10.1;Chapter Twelve: Use of 19F NMR Methods to Probe Conformational Heterogeneity and Dynamics of Exchange in Functional RNA M ...;292
10.1.1;1. Introduction;293
10.1.2;2. Methods;295
10.1.2.1;2.1. Sample design;295
10.1.2.2;2.2. Sample preparation;296
10.1.2.3;2.3. One-dimensional 19F experiments to identify the distribution of folds;296
10.1.2.4;2.4. Two-dimensional 19F-19F EXSY experiments to measure conformational exchange;297
10.1.2.5;2.5. Application to analysis of distribution and exchange in a bistable RNA stem loop;300
10.1.2.6;2.6. Application to analysis of distribution and exchange in a biologically significant system;301
10.1.3;3. Conclusion and Remarks;307
10.1.4;Acknowledgments;308
10.1.5;References;308
10.2;Chapter Thirteen: Site-Directed Spin-Labeling Strategies and Electron Paramagnetic Resonance Spectroscopy for Large Ribos ...;312
10.2.1;1. Techniques Used for Riboswitch Studies;315
10.2.1.1;1.1. Biochemical;315
10.2.1.2;1.2. Spectroscopy and labeling;317
10.2.1.2.1;1.2.1. EPR;317
10.2.1.3;1.3. Site-directed spin labeling;318
10.2.1.3.1;1.3.1. Labeling positions;318
10.2.1.3.2;1.3.2. Choice of spin label;320
10.2.1.3.3;1.3.3. SDSL for CW and pulsed EPR;321
10.2.1.3.4;1.3.4. Advantages/disadvantages;321
10.2.2;2. Ligation Methods for SDSL of Large Riboswitches;322
10.2.2.1;2.1. T4 DNA ligase;323
10.2.2.2;2.2. Considerations for SDSL and T4 DNA-mediated ligation of large riboswitches;324
10.2.2.2.1;2.2.1. Optimizing conditions;325
10.2.2.2.2;2.2.2. Protocol;326
10.2.2.2.2.1;2.2.2.1. Synthetic RNA preparations;326
10.2.2.2.2.1.1;2.2.2.1.1. Deprotection of synthetic RNA;327
10.2.2.2.2.1.2;2.2.2.1.2. Spin labeling of synthetic RNA;327
10.2.2.2.2.2;2.2.2.2. Transcribed RNA preparations;328
10.2.2.2.2.2.1;2.2.2.2.1. Dephosphorylation and monophosphorylation;328
10.2.2.2.2.3;2.2.2.3. Small- and large-scale ligations;328
10.2.2.2.2.3.1;2.2.2.3.1. Annealing;329
10.2.2.2.2.3.2;2.2.2.3.2. Ligation;329
10.2.2.2.2.3.3;2.2.2.3.3. Scaling up ligation reactions;330
10.2.2.2.2.4;2.2.2.4. Large-scale purification of ligation product;331
10.2.2.2.2.4.1;2.2.2.4.1. Large-scale PCA extraction;331
10.2.2.2.2.4.2;2.2.2.4.2. Large-scale ethanol precipitation;331
10.2.2.2.2.4.3;2.2.2.4.3. Purification by dPAGE;331
10.2.2.2.2.4.4;2.2.2.4.4. Sample preparation for CW-EPR;332
10.2.3;3. CW-EPR Spectral Analysis of Riboswitches;332
10.2.4;References;334
10.3;Chapter Fourteen: Using sm-FRET and Denaturants to Reveal Folding Landscapes;338
10.3.1;1. Introduction;339
10.3.2;2. Single-Molecule FRET: Technical Aspects;342
10.3.3;3. Riboswitch Structure and Biological Function;344
10.3.3.1;3.1. sm-FRET studies of the adenine aptamer under nondenaturing conditions;346
10.3.4;4. Combination of sm-FRET and Denaturants to Investigate Riboswitch Folding;348
10.3.4.1;4.1. Urea-induced perturbation of RNA folding: Ensemble studies;349
10.3.4.2;4.2. Urea-induced perturbation of RNA folding: Single-molecule studies;350
10.3.4.3;4.3. Technical considerations when combining sm-FRET and chemical denaturants;351
10.3.4.4;4.4. Urea-induced effects on the single-molecule dynamics of adenine aptamers;353
10.3.4.5;4.5. In situ cycling between Mg2+ and urea: A method to quantify the reversibility of chemical denaturation;354
10.3.4.6;4.6. Methods for comparing Mg2+-assisted folding and urea-induced unfolding;354
10.3.4.7;4.7. Influence of urea on the undocking rates: A method to quantify the ligand-induced stabilization of the aptamer domain;355
10.3.4.8;4.8. Influence of urea on the docking rates: A method to evaluate the rate-limiting step for folding;358
10.3.5;5. Summary and Prospects;362
10.3.6;References;362
10.4;Chapter Fifteen: Riboswitch Structure and Dynamics by smFRET Microscopy;368
10.4.1;1. Introduction;369
10.4.1.1;1.1. Single-molecule fluorescence resonance energy transfer;372
10.4.1.1.1;1.1.1. Advantages of single-molecule methods;372
10.4.1.1.2;1.1.2. Fluorescence resonance energy transfer;374
10.4.2;2. Methods;376
10.4.2.1;2.1. Labeling and purification of riboswitches;376
10.4.2.2;2.2. Preparation of quartz slides;379
10.4.2.3;2.3. Surface attachment and oxygen scavenging systems;381
10.4.2.4;2.4. smFRET using prism-based TIRF microscopy;382
10.4.2.5;2.5. Heat-annealing of riboswitch RNAs;383
10.4.3;3. Practical Experimental Considerations;384
10.4.4;4. Data Analysis;385
10.4.4.1;4.1. FRET histograms;386
10.4.4.2;4.2. Kinetic analysis;388
10.4.4.3;4.3. Cross-correlation analysis;390
10.4.5;5. Induced-Fit Versus Conformational Selection;390
10.4.6;6. Summary and Conclusions;393
10.4.7;Acknowledgments;393
10.4.8;References;393
10.5;Chapter Sixteen: Ribosome Structure and Dynamics by smFRET Microscopy;400
10.5.1;1. Introduction;401
10.5.2;2. Overview of Ribosome Structure and Function;402
10.5.3;3. Methodology;404
10.5.3.1;3.1. What is out there?;404
10.5.3.2;3.2. Why single-molecule approaches?;405
10.5.3.3;3.3. Why is the ribosome an ideal system for smFRET?;406
10.5.4;4. Ribosome Dynamics;409
10.5.4.1;4.1. Choosing a question;410
10.5.4.2;4.2. Choosing a dye;410
10.5.4.3;4.3. Using phylogenetic analysis and structural modeling to guide choice of labeling sites;411
10.5.4.4;4.4. Fluorescently labeling various translation components;414
10.5.4.4.1;4.4.1. tRNA labeling;414
10.5.4.4.2;4.4.2. Ribosome labeling;414
10.5.4.4.3;4.4.3. Translation factors labeling;416
10.5.4.5;4.5. Testing activity of purified translation components;416
10.5.4.5.1;4.5.1. Filter binding and puromycin reactivity assay;416
10.5.4.6;4.6. Assessing the spectroscopic properties of the labeled components;417
10.5.4.7;4.7. Ribosomal complex assembly;418
10.5.4.8;4.8. Immobilization schemes;418
10.5.4.9;4.9. Imaging;419
10.5.5;5. Data Acquisition;420
10.5.5.1;5.1. Selecting a camera;420
10.5.5.2;5.2. Signal to noise;420
10.5.5.2.1;5.2.1. Dark noise, readout noise, Poisson noise;420
10.5.5.2.2;5.2.2. Exposure time, QE, DR, gain;421
10.5.5.2.3;5.2.3. Binning;421
10.5.5.2.4;5.2.4. Nyquist theorem: Undersampling, oversampling;422
10.5.5.2.5;5.2.5. Bit depth;423
10.5.5.2.6;5.2.6. Improving temporal resolution;423
10.5.5.3;5.3. Acquisition;423
10.5.5.3.1;5.3.1. Analysis algorithms;424
10.5.6;6. Building and Verifying Histograms, Normalization, Gaussian Fitting;425
10.5.7;7. Future Directions;425
10.5.8;References;426
10.6;Chapter Seventeen: Unraveling the Thermodynamics and Kinetics of RNA Assembly: Surface Plasmon Resonance, Isothermal Titr...;432
10.6.1;1. The RNA Folding Problem and Assembly of RNA Tertiary Structure;433
10.6.2;2. Practical Aspects of Biophysical Studies of RNA Assembly;435
10.6.2.1;2.1. RNA assembly: Specification and control of ionic conditions;435
10.6.2.1.1;2.1.1. Protocol 1: Large-scale RNA purification and buffer exchange via dialysis;437
10.6.2.2;2.2. RNA assembly: Binding parameters and choice of methodology;438
10.6.2.3;2.3. RNA assembly: Analytical aspects and experimental design;439
10.6.3;3. Specific Measurements of Ion-Driven RNA Assembly;442
10.6.3.1;3.1. Ion dependence of assembly: CD;442
10.6.3.1.1;3.1.1. Protocol 2;444
10.6.3.2;3.2. Rates of intermolecular assembly: SPR;446
10.6.3.2.1;3.2.1. Protocol 3;447
10.6.3.3;3.3. Thermodynamics of RNA assembly: ITC;449
10.6.3.3.1;3.3.1. ITC experimental design;451
10.6.3.3.2;3.3.2. Experimental design: Buffer match;453
10.6.4;4. Concluding Remarks;454
10.6.5;Acknowledgments;454
10.6.6;References;454
11;Part V: Ligand Interactions;458
11.1;Chapter Eighteen: ITC Analysis of Ligand Binding to PreQ1 Riboswitches;460
11.1.1;1. Introduction;461
11.1.1.1;1.1. Information content in an ITC experiment;463
11.1.2;2. Experimental Procedures for ITC;464
11.1.2.1;2.1. Assessing the feasibility of ITC experiments;464
11.1.2.2;2.2. Instrumentation, materials, and solutions for ITC;466
11.1.2.3;2.3. RNA and ligand preparation;467
11.1.2.4;2.4. The isothermal titration calorimetry experiment;468
11.1.2.5;2.5. ITC data analysis;469
11.1.2.6;2.6. Manual adjustment of the baseline;471
11.1.2.7;2.7. Publishing ITC results and representative analysis;471
11.1.3;Acknowledgments;473
11.1.4;References;474
11.2;Chapter Nineteen: Facile Characterization of Aptamer Kinetic and Equilibrium Binding Properties Using Surface Plasmon Res...;476
11.2.1;1. Introduction;477
11.2.2;2. Materials;478
11.2.2.1;2.1. Instrumentation;478
11.2.2.2;2.2. Sensor surface immobilization;478
11.2.2.3;2.3. Aptamer binding assay;479
11.2.3;3. Sensor Surface Immobilization;480
11.2.3.1;3.1. Pre-concentration assay;480
11.2.3.2;3.2. DNA linker immobilization;482
11.2.4;4. Characterization of Aptamer Binding Properties;483
11.2.4.1;4.1. Aptamer design and preparation;483
11.2.4.2;4.2. Startup cycles;484
11.2.4.3;4.3. Aptamer binding assay;486
11.2.4.4;4.4. Analysis of aptamer binding properties;488
11.2.5;5. Conclusion;490
11.2.6;Acknowledgments;491
11.2.7;References;491
11.3;Chapter Twenty: The AdoCbl-Riboswitch Interaction Investigated by In-Line Probing and Surface Plasmon Resonance Spectrosc...;492
11.3.1;1. Introduction;493
11.3.2;2. In-Line Probing Experiments;496
11.3.2.1;2.1. Mechanism of the in-line probing reaction;496
11.3.2.2;2.2. Performing an in-line probing experiment;497
11.3.2.2.1;2.2.1. Overview and general remarks;497
11.3.2.2.2;2.2.2. Equipment for in-line probing;497
11.3.2.2.3;2.2.3. Buffers and solutions for in-line probing;498
11.3.2.2.4;2.2.4. In-line probing reaction and PAGE analysis;498
11.3.2.2.5;2.2.5. Data analysis and calculation of KD values;499
11.3.3;3. SPR Spectroscopy;502
11.3.3.1;3.1. The method of SPR;502
11.3.3.2;3.2. Practical example: Studying the AdoCbl-btuB riboswitch interaction by SPR;503
11.3.3.2.1;3.2.1. Overview and general remarks;503
11.3.3.2.2;3.2.2. Equipments for SPR;504
11.3.3.2.3;3.2.3. Buffers and solutions for SPR;504
11.3.3.2.4;3.2.4. Sample preparation;505
11.3.3.2.5;3.2.5. Immobilization of the RNA on sensor surface;505
11.3.3.2.6;3.2.6. SPR measurements;507
11.3.3.2.7;3.2.7. Data analysis;509
11.3.4;4. Conclusion;509
11.3.5;Acknowledgments;510
11.3.6;References;510
11.4;Chapter Twenty-One: Assessing RNA Interactions with Proteins by DRaCALA;514
11.4.1;1. Introduction;515
11.4.2;2. DRaCALA-Based Detection of Protein-Ligand Interactions;516
11.4.3;3. Principle of DRaCALA;518
11.4.4;4. Determination of Fraction Bound by DRaCALA;518
11.4.5;5. Steps for Performing DRaCALA to Detect Protein Interaction With RNA;520
11.4.5.1;5.1. Procedure: Preparation of expression vector;520
11.4.5.1.1;5.1.1. Reagents;520
11.4.5.1.2;5.1.2. Method;520
11.4.5.2;5.2. Procedure: Preparation of whole cell lysates;521
11.4.5.2.1;5.2.1. Reagents;521
11.4.5.2.2;5.2.2. Method;521
11.4.5.3;5.3. Procedure: Template generation;522
11.4.5.3.1;5.3.1. Reagents;522
11.4.5.3.2;5.3.2. Method;522
11.4.5.3.3;5.3.3. Consideration for template generation;523
11.4.5.4;5.4. Procedure: In vitro transcription of RNA;523
11.4.5.4.1;5.4.1. Reagents;523
11.4.5.4.2;5.4.2. Method;524
11.4.5.5;5.5. Procedure: 5-end labeling of RNA;525
11.4.5.5.1;5.5.1. Reagents;525
11.4.5.5.2;5.5.2. Method: AnP treatment to remove 5-triphosphate;526
11.4.5.5.3;5.5.3. Method: 5-end labeling of RsmY and RsmZ;527
11.4.5.5.4;5.5.4. Consideration for labeling of RNA ligand;528
11.4.5.6;5.6. Procedure: Determining protein-RNA interaction;528
11.4.5.6.1;5.6.1. Reagents;528
11.4.5.6.2;5.6.2. Method: Binding reaction and spotting of DRaCALA spots;529
11.4.5.7;5.7. Procedure: Determining relative affinity;532
11.4.5.7.1;5.7.1. Method;532
11.4.5.8;5.8. Procedure: Determining specificity of binding through competition;533
11.4.5.8.1;5.8.1. Method;533
11.4.6;6. CsrA Binds Specifically to RsmY and RsmZ;534
11.4.7;7. Other Modifications of DRaCALA for RNA-Protein Interactions;535
11.4.8;References;536
12;Author Index;538
13;Subject Index;560
14;Color Plate;572

Leseproben

Preface
Donald H. Burke-Aguero The early years of the twenty-first century have seen an explosion of interest in the diverse capabilities of RNA. Riboswitches capture the excitement and promise of this field. They are structurally dynamic, they sense and respond to specific molecular partners, their occupancy states governs gene regulatory decisions, and they can be engineered to reprogram gene regulatory circuitry. Importantly, many of the experimental and theoretical tools that have been used to study riboswitches can also be applied to other RNAs, and tools developed for studies of other RNAs can be applied to riboswitches. These two volumes (Methods in Enzymology 549 and 550) include 40 contributions that outline cutting-edge methods representing a wide spectrum of research questions and scientific themes. The first volume emphasizes natural riboswitches, from their discovery to assessment of their structures and functions. The second volume shifts the focus to applying riboswitches as tools for a variety of applications and as targets for inhibition by potential new antibacterial compounds. A third volume (Methods in Enzymology 553) will appear shortly after these two focusing on computational methods for predicting and evaluating dynamic RNA structures. Although the chapters are organized into discrete themes, many cut across thematic boundaries by weaving together diverse methodological solutions and several of the chapters could fit comfortably into more than one section. Volume 1
Riboswitch discovery. In the early days of the riboswitch field, new riboswitches were discovered at a frenetic pace, often by comparing large sets of bacterial genomes. While that approach continues to identify new members of known riboswitch families, the pace has slowed, and new discovery methods are needed. The series begins with two chapters outlining new methods that utilize informatics approaches in combination either with RNASeq and genome-wide methods (Rosinski-Chupin) or with in vitro selection (Ho) to discover new natural riboswitches. Sample preparation. Any effort to characterize purified, functional RNAs will only be as good as the corresponding sample preparations. Therefore, the next five chapters are dedicated to methods for the synthesis and preparation of large RNAs. Three groups exploit specialty nucleic acids with functionalities of their own. The first chapter in this set describes the use of cotranscribed aptamer affinity tags that are removed by activatable self-cleavage (Di Tomasso). This is followed by methods for using catalytic deoxyribozyme ligases to assemble large RNAs from synthetic fragments, some of which carry site-specific spin labels for electron paired resonance studies (Wawrzyniak-Turek). The third chapter in this set describes the combined use of aminoacyl transferase ribozymes and chemical protection to generate charged tRNAs on a large scale (Zhang). These are followed by two chapters that integrate organic chemical methods with improved enzymology to produce photocleavable biotinylated guanosine that incorporates at the 5' end of in vitro transcripts (Luo) and large quantities of selectively 13C/15N-labeled RNA in previously unattainable labeling patterns for improved spectroscopic analysis (Alvarado). Structure and function. The biochemical functions of riboswitches are inextricably linked with their three-dimensional structures. The next several chapters, therefore, provide methods for evaluating riboswitch structure and function. Updated protocols are provided for the widely utilized SHAPE method of structural probing, along with details of how to implement new software for data interpretation (Rice). It is well recognized that structural context can perturb pKa values within RNA and DNA; hence, the next chapter details how to measure them without falling into traps of oversimplifying the underlying molecular processes (Taplyal). The next chapter provides methods for obtaining appropriate crystals for ligand–RNA complexes, with emphasis on fragment-bound TPP riboswitches (Warner). This section ends with a detailed description of experimental and analytical methods for using small-angle X-ray scattering to define RNA conformations in solution (Reyes). Conformational dynamics. Spectroscopic methods are ideal for following riboswitch conformational dynamics in real time. The first two chapters of this section describe site-specific incorporation of spectroscopic labels and their use in addressing specific question, first with 19F NMR to probe conformational exchange (Zhao) and then with spin-label probes for electron paramagnetic resonance spectroscopy of large RNAs (Esquiaqui). Single-molecule methods such as smFRET have become a staple of modern biophysical analysis. Three chapters provide detailed guidance on many facets of smFRET, from sample preparation, data acquisition, and analysis to explorations of folding landscapes (Shaw, Suddala, and Shebl). The last chapter of this section describes how to integrate surface plasmon resonance (SPR), isothermal titration calorimetry, and circular dichroism to examine tertiary docking (Hoogstraten). Ligand interactions. One of the most important characteristics of riboswitches is their ability to sense the presence of specific metabolites by forming bound molecular complexes. Isothermal titration calorimetry is one of the most powerful methods for evaluating the energetics of RNA–small molecule complexes (Wedekind). SPR is another powerful tool for characterizing aptamer kinetic and equilibrium binding properties and is detailed in two chapters (Chang and Schaffer). Finally, an innovative and relatively new technique known as DRaCALA is described in the last chapter of the first volume (Patel). Volume 2
The second volume in this series takes a different perspective on riboswitches. Specifically, now that nature has shown us that RNA modules can sense metabolites and report on them, how can we take advantage of that ability to engineer new properties into cells and biochemical systems? Necessarily, this volume takes a much broader view of riboswitches than those found in nature, encompassing ligand-responsive transcriptional and translational modules, ribozymes, sensors, and modules that induce fluorescence in a fluorophore upon formation of the bound complex. It encompasses Synthetic Biology applications as tools to understand normal biological processes, and as tools to reprogram metabolite flux in workhorse organisms. Finally, it comes full circle by screening small-molecule libraries for inhibitors of natural riboswitches. In short, this second volume details methods at the cutting edge of the translational science of riboswitches. Artificial riboswitches. The first six chapters of the second volume provide methods for several approaches to construct and optimize artificial riboswitches. There has been substantial progress toward designing artificial riboswitches from scratch, especially when guided by experimental validation (Moerl). A contrasting approach uses in vitro selection/evolution to obtain ligand-responsive ligase ribozymes from highly diverse starting populations (Olea), or to reshape and reprogram the ligand-binding and expression platforms of natural riboswitches (Batey). The next chapter presents methods for optimizing signal transduction, since regulation sometimes benefits from maximizing suppression of basal expression in the OFF state and sometimes from maximizing expression in the ON state (Goodson). The next two chapters address optimization in two very different cell-free systems, first using coupled transcription–translation to optimize a ligand-responsive self-cleaving ribozyme, or “aptazyme” (Ichihashi), and then taking advantage of a eukaryotic mechanism by which ribosomes “shunt” past certain secondary structures, which can be stabilized to increase shunting efficiency by binding to the analyte ligand (Ogawa). Ligand-responsive fluorescent sensors. There has been longstanding interest in coupling the binding of ligands to RNA with the emission of light. One such system is that of the recently described Spinach (and Spinach2) aptamer mimics of green fluorescent protein, which are the focus of the next five chapters, each in a different system. The first chapter in this section, from the lab that discovered and first described the Spinach system, presents methods for using it to image intracellular RNA in mammalian cells (Strack). The next two chapters describe how to use these modules in bacterial cells, first as intracellular sensors of intracellular cyclic dinucleotide levels (Kellenberger) and then for simultaneous and independent monitoring of mRNA and protein levels (Pothoulakis). The next chapter takes this same question into solution and into vesicle-based artificial cells (van Nies). The fifth chapter in this section couples sensing of oligonucleotide “ligands” with Spinach2 output in real time for sequence-specific target quantitation and potential point-of-care applications (Bhadra). Synthetic biology: Conditional control of gene expression. The third section of this volume lays out several methods for using artificial or natural riboswitches to study gene function. This has proven to be a powerful tool in organisms for which limited genetic tools are available, such as the intracellular pathogen Mycobacteria (Van Vlack), as well as in more readily manipulated, nonpathogenic bacteria such as Streptomyces coelicolor (Rudolph). Eukaryotes...