Riboswitches as Targets and Tools

1. Auflage 2015
ISBN: 978-0-12-801336-6
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark

E-Book, Englisch, Band Volume 550, 444 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 riboswitches as targets and tools and contains sections on such topics as constructing and optimizing artificial riboswitches, live cell imaging and intracellular sensors with artificial riboswitches, conditional control of gene expression with artificial riboswitches, using artificial riboswitches for protein evolution and pathway optimization, and anti-riboswitch drug screens. - Continues the legacy of this premier serial with quality chapters authored by leaders in the field - Covers research methods in riboswitches as targets and tools - Contains sections on such topics as constructing and optimizing artificial riboswitches, synthetic biology: live cell imaging and intracellular sensors with artificial riboswitches, synthetic biology: conditional control of gene expression with artificial riboswitches, synthetic biology: using artificial riboswitches for protein evolution and pathway optimization, anti-riboswitches drug screens

Autoren/Hrsg.

Inhaltsverzeichnis

1;Front Cover;1
2;Riboswitches as Targets and Tools;4
3;Copyright;5
4;Contents;6
5;Contributors;12
6;Preface;16
6.1;Volume 1;16
6.2;Volume 2;18
7;Chapter 1: Design of Transcription Regulating Riboswitches;22
7.1;1. Introduction;23
7.2;2. Computational Design of RNA Structures;26
7.2.1;2.1. The inverse folding problem;26
7.2.2;2.2. Designing multi-stable RNAs;30
7.2.3;2.3. Modeling external triggers;31
7.2.4;2.4. Current limitation of design software;33
7.3;3. Experimental Evaluation of Designed RNA Structures;36
7.3.1;3.1. Considerations on candidate selection and cloning procedures;36
7.3.2;3.2. Further characterization and current limitations;38
7.4;4. Concluding Remarks;39
7.5;References;40
8;Chapter 2: Ligand-Dependent Exponential Amplification of Self-Replicating RNA Enzymes;44
8.1;1. Introduction;45
8.2;2. Exponential Amplification of RNA Enzymes;46
8.2.1;2.1. Materials;47
8.2.2;2.2. Procedure for RNA self-replication;48
8.3;3. Ligand-Dependent Exponential Amplification;49
8.3.1;3.1. Procedure for quantitative ligand detection;50
8.3.2;3.2. Multiplexed ligand detection;51
8.3.3;3.3. Coupling ligand recognition to ligand-independent amplification;52
8.3.4;3.4. Procedure for coupled amplification;53
8.4;4. Nuclease-Resistant Autocatalytic Aptazymes;56
8.5;5. Real-Time Fluorescence Assays;57
8.6;6. Conclusions;59
8.7;Acknowledgments;59
8.8;References;59
9;Chapter 3: Design of Modular ``Plug-and-Play´´ Expression Platforms Derived from Natural Riboswitches for Engineering Nov...;62
9.1;1. Introduction;63
9.2;2. Design of Riboswitch Modules;67
9.2.1;2.1. Design strategy;67
9.2.2;2.2. Design optimization;75
9.3;3. Analysis of Riboswitch Activity Using an In Vitro Single-Turnover Transcription Assay;77
9.3.1;3.1. Template construction;77
9.3.1.1;3.1.1. Overlapping extension PCR;79
9.3.1.2;3.1.2. Purification of the template;80
9.3.2;3.2. Single-turnover in vitro transcription assay;81
9.4;4. Cell-Based GFP Reporter Assay;83
9.4.1;4.1. Reporter design;84
9.4.2;4.2. Protocol;84
9.4.3;4.3. Other considerations using in vivo reporters;87
9.5;5. Concluding Remarks;88
9.6;Acknowledgment;89
9.7;References;89
10;Chapter 4: Integrating and Amplifying Signal from Riboswitch Biosensors;94
10.1;1. Introduction;95
10.1.1;1.1. Biological circuits;95
10.2;2. Riboswitch Signal Integration;97
10.2.1;2.1. Design;97
10.2.1.1;2.1.1. AND gate;97
10.2.1.2;2.1.2. Selection of riboswitches;98
10.2.1.3;2.1.3. Using plasmid backbones available from the Registry of Standard Biological Parts;100
10.2.2;2.2. Build;100
10.2.2.1;2.2.1. Materials for construction of a riboswitch-based AND logic gate;100
10.2.2.2;2.2.2. Methods to construct pANDrs;102
10.2.3;2.3. Test;102
10.3;3. Riboswitch Signal Amplification Using Biological Circuitry;103
10.3.1;3.1. Design;103
10.3.2;3.2. Build;105
10.3.2.1;3.2.1. Materials for the construction of an amplification circuit;105
10.3.2.2;3.2.2. Methods to construct an amplification circuit, pAMPv1;106
10.3.2.2.1;3.2.2.1. PLas_RBS34(strong)_GFPa1_Terminator_Weak RBS_LasI +/-degradation tag;106
10.3.3;3.3. Test;106
10.3.4;3.4. Redesign and build;106
10.3.4.1;3.4.1. Methods to construct amplification circuit version 2;109
10.3.4.1.1;3.4.1.1. pAMPv2.1: PLas_RBS 34 (strong)_GFPa1_Terminator_RBS 33 (very weak)_RhlILVA;109
10.3.4.1.2;3.4.1.2. pAMPv2.2: PRhl_RBS 34 (strong)_GFPa1_Terminator_RBS 33 (very weak)_LasILVA;109
10.3.5;3.5. Test;109
10.3.5.1;3.5.1. Fluorescence activation;109
10.3.5.2;3.5.2. Signal progression;109
10.4;Acknowledgments;110
10.5;References;110
11;Chapter 5: Simple Identification of Two Causes of Noise in an Aptazyme System by Monitoring Cell-Free Transcription;114
11.1;1. Theory;115
11.2;2. Equipment;117
11.3;3. Materials;117
11.4;4. Solutions and Buffers;118
11.5;5. Protocol;119
11.5.1;5.1. Duration;119
11.5.2;5.2. Preparation;119
11.5.3;5.3. Caution;120
11.6;6. Step 1: Cell-Free Transcription-Translation and Fluorescence Monitoring;120
11.6.1;6.1. Overview;120
11.6.2;6.2. Duration;120
11.6.3;6.3. Tip;121
11.6.4;6.4. Tip;122
11.7;7. Step 2: Data Analysis;122
11.7.1;7.1. Overview;122
11.7.2;7.2. Duration;123
11.7.3;7.3. Tip;124
11.7.4;7.4. Tip;124
11.8;8. Step 3: (Optional) Quantification of the Intermediate RNAs;125
11.8.1;8.1. Overview;125
11.8.2;8.2. Duration;125
11.8.3;8.3. Tip;127
11.8.4;8.4. Tip;128
11.9;References;128
12;Chapter 6: Engineering of Ribosomal Shunt-Modulating Eukaryotic ON Riboswitches by Using a Cell-Free Translation System;130
12.1;1. Introduction;131
12.2;2. A Eukaryotic Translation Mechanism Requiring a Rigid mRNA Structure for Ribosomal Progression;133
12.3;3. Choice of a Translation System for Engineering Artificial Riboswitches;135
12.4;4. In Vitro-Selected Aptamer for Ribosomal Shunt-Modulating Riboswitches;136
12.5;5. How to Implant the Selected Aptamer into mRNA;137
12.6;6. General Design of mRNAs with Ribosomal Shunt-Modulating Riboswitches;140
12.6.1;6.1. 5 Untranslated region;140
12.6.2;6.2. Short open reading frame;140
12.6.3;6.3. Takeoff site;141
12.6.4;6.4. Aptamer-Ks conjugate;141
12.6.5;6.5. Landing site;141
12.6.6;6.6. Downstream open reading frame;141
12.6.7;6.7. 3 Untranslated region;142
12.7;7. Experiments;142
12.7.1;7.1. Construction of DNA templates for mRNAs;142
12.7.2;7.2. In vitro transcription;144
12.7.3;7.3. In vitro translation in WGE;144
12.7.4;7.4. Characterization of riboswitches;145
12.8;8. Conclusion;146
12.9;Acknowledgments;147
12.10;References;147
13;Chapter 7: Live-Cell Imaging of Mammalian RNAs with Spinach2;150
13.1;1. Introduction;150
13.2;2. Developing Spinach, an RNA Mimic of GFP;153
13.3;3. Imaging with Spinach2, a Superfolding Variant of Spinach;155
13.3.1;3.1. Tagging an RNA of interest with Spinach2;156
13.3.2;3.2. Testing a tagged RNA for Spinach2 fluorescence in vitro;156
13.3.3;3.3. Expressing 5S RNA in HEK 293-T cells;158
13.3.4;3.4. Expressing CGG60-Spinach2 in COS-7 cells;159
13.4;4. Fluorescence Imaging of Spinach2-Tagged RNAs;161
13.4.1;4.1. Imaging 5S-Spinach2;162
13.4.2;4.2. Imaging CGG60-Spinach2;163
13.5;5. Imaging Other RNAs Using Spinach2;165
13.6;Acknowledgments;165
13.7;References;165
14;Chapter 8: In Vitro Analysis of Riboswitch-Spinach Aptamer Fusions as Metabolite-Sensing Fluorescent Biosensors;168
14.1;1. Introduction;169
14.1.1;1.1. General equipment;171
14.1.2;1.2. General materials;171
14.2;2. Design and Preparation of an RNA-Based Fluorescent Biosensor;172
14.2.1;2.1. Preparation of DNA templates of riboswitch-Spinach aptamer fusion;173
14.2.1.1;2.1.1. Equipment and materials;175
14.2.1.2;2.1.2. Procedure for generating DNA template for transcription;175
14.2.2;2.2. Preparation of RNAs by in vitro transcription;175
14.2.2.1;2.2.1. Materials and equipment;175
14.2.2.2;2.2.2. Procedures;178
14.3;3. Determination of Ligand Selectivity and Affinity of Biosensor by Fluorescence Activation;181
14.3.1;3.1. Determination of ligand selectivity;181
14.3.1.1;3.1.1. Additional materials and equipment;181
14.3.1.2;3.1.2. Procedures;182
14.3.2;3.2. Determination of ligand affinity;185
14.4;4. Determination of Binding Kinetics of Biosensor;186
14.4.1;4.1. Determination of activation rate;188
14.4.1.1;4.2. Determination of deactivation rate;189
14.4.1.1.1;4.2.1. Additional materials and equipment;189
14.4.1.1.2;4.2.2. Procedure;190
14.5;References;192
15;Chapter 9: Using Spinach Aptamer to Correlate mRNA and Protein Levels in Escherichia coli;194
15.1;1. Introduction;195
15.2;2. Parts Selection and Plasmid Construction;196
15.3;3. E. coli Strain Selection;197
15.4;4. Culturing and Inducing E. coli Cells;199
15.5;5. Correlating mRNA and Protein Production Using Flow Cytometry;199
15.5.1;5.1. Prepping cells for analysis;200
15.5.2;5.2. Calibrating and setting up the flow cytometer;200
15.5.3;5.3. Analysis of flow data and presentation;201
15.6;6. Correlating mRNA and Protein Production Using Fluorescence Microscopy;201
15.6.1;6.1. Agarose pads;203
15.6.2;6.2. Prepping cells for analysis;203
15.6.3;6.3. Setting up the fluorescence microscope;204
15.6.4;6.4. Image analysis;205
15.7;7. Summary;205
15.8;Acknowledgments;206
15.9;References;206
16;Chapter 10: Monitoring mRNA and Protein Levels in Bulk and in Model Vesicle-Based Artificial Cells;208
16.1;1. Introduction;209
16.2;2. The ``Spinach Technology´´ for Combined Detection of mRNA and Protein in Cell-Free Expression Systems;211
16.2.1;2.1. Lighting up RNA with Spinach;211
16.2.2;2.2. Synthesis of DFHBI;212
16.2.3;2.3. Design and preparation of the DNA templates;213
16.2.4;2.4. In vitro transcription-translation with PUREfrex;214
16.2.5;2.5. Kinetics measurements by spectrofluorometry;215
16.2.6;2.6. Characterization of improved Spinach fluorescence in the PURE system;215
16.2.7;2.7. Orthogonal detection of synthesized mRNA-Spinach and protein;218
16.3;3. Quantifying the Levels of mRNA and Protein Synthesized in PURE System Bulk Reactions;219
16.3.1;3.1. Overall workflow;219
16.3.2;3.2. Preparation of reference RNA and purification;220
16.3.3;3.3. Gel analysis of mRNA concentration;220
16.3.4;3.4. Real-time quantitative PCR analysis;222
16.3.5;3.5. mRNA quantification from Spinach fluorescence of reference RNA;222
16.3.6;3.6. Calculating mYFP concentration by fluorescence correlation spectroscopy and absorbance measurements;223
16.3.6.1;3.6.1. Fluorescence correlation spectroscopy setup;223
16.3.6.2;3.6.2. Calibration of the detection volume;223
16.3.6.3;3.6.3. Measuring the concentration of synthesized mYFP;224
16.3.6.4;3.6.4. Converting mYFP fluorescence intensity into concentration;225
16.3.6.5;3.6.5. Absorbance measurements;226
16.3.6.6;3.6.6. FCS versus absorbance measurements;226
16.3.7;3.7. Quantitative analysis of mRNA and protein concentration versus time;226
16.4;4. Detecting Gene Expression Inside Semipermeable Liposomes;228
16.4.1;4.1. Preparation of lipid film-coated beads;229
16.4.2;4.2. Liposome formation and encapsulation of the biosynthesis machinery;229
16.4.3;4.3. Surface functionalization, liposome immobilization, and triggering of gene expression;230
16.4.4;4.4. Visualizing liposomes with fluorescence microscopy;231
16.4.5;4.5. Factors influencing the levels of mRNA and protein produced in liposomes;231
16.5;5. Conclusion and Outlook;232
16.6;Acknowledgments;233
16.7;References;233
17;Chapter 11: Design, Synthesis, and Application of Spinach Molecular Beacons Triggered by Strand Displacement;236
17.1;1. Introduction;237
17.2;2. How to Engineer Spinach Molecular Beacons Triggered by Toehold-Mediated Strand Displacement;239
17.2.1;2.1. Engineering conformations and sequence modules for Spinach beacons;239
17.2.2;2.2. Using NUPACK and KineFold for design;244
17.2.3;2.3. Promoters for Spinach.ST expression;246
17.2.4;2.4. Stabilization of Spinach.ST within a tRNA scaffold;247
17.2.5;2.5. Programming triggers;247
17.2.5.1;2.5.1. Designing triggers to discriminate single-nucleotide mismatches;248
17.2.5.2;2.5.2. Designing triggers to function in the context of nucleic acid circuits;248
17.2.5.3;2.5.3. Associative toehold triggers;251
17.2.6;2.6. Spinach.ST function within larger RNA contexts;252
17.3;3. How to Synthesize Spinach.ST Molecular Beacons Enzymatically;254
17.3.1;3.1. DNA oligonucleotides;254
17.3.2;3.2. How to generate transcription templates;254
17.4;4. How to Perform Functional Assays of Spinach.ST Molecular Beacons;255
17.4.1;4.1. In vitro transcription reactions;255
17.4.2;4.2. Reactions with added trigger and control sequences;256
17.4.3;4.3. Real-time cotranscriptional functional assays with Spinach.ST;257
17.5;5. Application: Real-Time Spinach.ST-Based Detection of NASBA;258
17.5.1;5.1. Including Spinach.ST trigger components in NASBA primers;258
17.5.2;5.2. Detecting NASBA amplification with Spinach.ST;264
17.6;6. Conclusions;265
17.7;Acknowledgments;266
17.8;References;266
18;Chapter 12: Using Riboswitches to Regulate Gene Expression and Define Gene Function in Mycobacteria;272
18.1;1. Introduction;273
18.2;2. Riboswitch Reporter Assays;274
18.2.1;2.1. Construction of promoter-riboswitch GFP or ß-galactosidase reporter plasmids;279
18.2.2;2.2. GFP fluorescence endpoint assay;280
18.2.2.1;2.2.1. Note on culturing bacteria in multi-well plates;281
18.2.3;2.3. GFP flow cytometry;281
18.2.4;2.4. ß-Galactosidase activity endpoint assay;282
18.3;3. Construction of Recombinant Strains with Riboswitch-Regulated Genes;283
18.4;4. Induction of Mycobacterial Genes in Infected Host Cells;284
18.5;Acknowledgments;285
18.6;References;285
19;Chapter 13: Controlling Expression of Genes in the Unicellular Alga Chlamydomonas reinhardtii with a Vitamin-Repressible ...;288
19.1;1. Introduction;289
19.2;2. Design of the Repressible Riboswitch System Acting on Chloroplast Genes;290
19.3;3. Methods;295
19.3.1;3.1. Growth conditions;295
19.3.2;3.2. Nuclear transformation;296
19.3.3;3.3. Chloroplast transformation;297
19.3.4;3.4. Screening for essential chloroplast genes;298
19.3.5;3.5. Effect of vitamins;299
19.4;4. Conclusions and Perspectives;299
19.5;Acknowledgments;300
19.6;References;300
20;Chapter 14: Conditional Control of Gene Expression by Synthetic Riboswitches in Streptomyces coelicolor;304
20.1;1. Introduction;305
20.2;2. Construction of Riboswitch-Controlled Expression Systems;307
20.2.1;2.1. Vector;307
20.2.2;2.2. Riboswitch design;308
20.2.3;2.3. Genetic manipulations in S. coelicolor;310
20.3;3. Measurement of Riboswitch Activity;310
20.3.1;3.1. ß-Glucuronidase measurement;310
20.3.2;3.2. Detection on agar plates;310
20.3.3;3.3. Measurement in liquid culture;311
20.4;4. Characterization of Riboswitch-Controlled Gene Expression;311
20.4.1;4.1. Dynamic range of regulation can be adjusted by appropriate promoter-riboswitch pairing;311
20.4.2;4.2. Dose dependence of riboswitch regulation;316
20.4.3;4.3. Assessing kinetics of induction and repression;317
20.5;5. Conclusion;317
20.6;Acknowledgments;318
20.7;References;319
21;Chapter 15: Engineering of Ribozyme-Based Aminoglycoside Switches of Gene Expression by In Vivo Genetic Selection in Sacc...;322
21.1;1. Theory;323
21.2;2. Equipment and Material;327
21.2.1;2.1. Molecular subcloning of the aptazyme library and propagation into Escherichia coli;328
21.2.2;2.2. Selection and hit identification of the aptazyme library;330
21.3;3. Protocol;331
21.3.1;3.1. Aptazyme library construction;331
21.3.2;3.2. Transformation of the aptazyme library into E. coli XL10 gold;335
21.3.3;3.3. Generation of electrocompetent yeast cells;336
21.3.4;3.4. Selection and screening of the yeast aptazyme library and ``hit´´ identification;337
21.4;References;339
22;Chapter 16: Kinetic Folding Design of Aptazyme-Regulated Expression Devices as Riboswitches for Metabolic Engineering;342
22.1;1. Introduction;343
22.2;2. In Vitro Characterization;345
22.2.1;2.1. Materials and equipment;347
22.2.2;2.2. Methods;348
22.2.2.1;2.2.1. DNA oligo purification;348
22.2.2.2;2.2.2. Ethanol precipitation;350
22.2.2.3;2.2.3. Cotranscriptional cleavage analysis;350
22.3;3. In Silico Transcript Design;352
22.3.1;3.1. System and submission guidelines;354
22.3.2;3.2. Computational methods;355
22.4;4. In Vivo Validation;357
22.4.1;4.1. Equipment and reagents;358
22.4.2;4.2. Methods;358
22.5;5. Future Directions;359
22.6;Acknowledgment;360
22.7;References;360
23;Chapter 17: Riboselector: Riboswitch-Based Synthetic Selection Device to Expedite Evolution of Metabolite-Producing Micro ...;362
23.1;1. Introduction;363
23.2;2. Materials;365
23.2.1;2.1. Equipment;365
23.2.2;2.2. Materials;365
23.2.3;2.3. Oligonucleotides;365
23.3;3. Construction and Validation of Riboselector: Riboswitch-Based Synthetic Selection Devices;365
23.3.1;3.1. Riboselector based on a natural riboswitch;365
23.3.1.1;3.1.1. Construction of a device for reporting its operation;370
23.3.1.2;3.1.2. Characterization of the constructed reporting device;371
23.3.2;3.2. Riboselector based on an artificial riboswitch from synthetic aptamer;372
23.3.2.1;3.2.1. Library generation for selection of synthetic device;372
23.3.2.2;3.2.2. Iterative positive and negative selection of synthetic device;373
23.3.2.3;3.2.3. Characterization of potential synthetic device;375
23.3.3;3.3. Characterization and validation of the constructed Riboselector;375
23.3.3.1;3.3.1. Growth rate characterization with Riboselector under selective pressure;375
23.3.3.2;3.3.2. Growth competition with Riboselector under selective pressure;376
23.4;4. Application of Riboselector for Pathway Engineering;376
23.4.1;4.1. Metabolic pathway engineering for phenotypic diversification;376
23.4.1.1;4.1.1. Pathway engineering to enhance lysine production;378
23.4.1.2;4.1.2. Library generation for optimization of ppc expression level;379
23.4.2;4.2. Pathway optimization using synthetic selection device;380
23.4.2.1;4.2.1. Setting an appropriate selection pressure by measuring growth rates;380
23.4.2.2;4.2.2. Enrichment experiment and analysis;380
23.5;5. Concluding Remarks;381
23.6;Acknowledgments;381
23.7;References;382
24;Chapter 18: Fluorescence Assays for Monitoring RNA-Ligand Interactions and Riboswitch-Targeted Drug Discovery Screening;384
24.1;1. Introduction;385
24.1.1;1.1. Noncoding RNAs and drug discovery;385
24.1.2;1.2. Screening cascade for RNA-targeted drug discovery;387
24.2;2. General Considerations;390
24.2.1;2.1. Equipment;390
24.2.2;2.2. Setup and optimization;390
24.2.2.1;2.2.1. Microplate setup and spectrometer settings;390
24.2.2.2;2.2.2. Ligand fluorescence;391
24.2.3;2.3. Pipetting and assay preparation;391
24.2.4;2.4. Materials and reagents;392
24.3;3. Example Protocols;394
24.3.1;3.1. 1 Screening assays;394
24.3.1.1;3.1.1. Steady-state fluorescence assay: Ligand-antiterminator RNA binding assay;394
24.3.1.1.1;3.1.1.1. Materials and reagents;394
24.3.1.1.2;3.1.1.2. Reagent plate preparation;395
24.3.1.1.3;3.1.1.3. Mixing plate preparation;395
24.3.1.1.4;3.1.1.4. Assay plate preparation and data acquisition;396
24.3.1.1.5;3.1.1.5. Analysis;396
24.3.1.2;3.1.2. Fluorescence anisotropy assay: Ligand-induced tRNA-antiterminator complex disruption assay;397
24.3.1.2.1;3.1.2.1. Materials and reagents;397
24.3.1.2.2;3.1.2.2. Reagent plate preparation;398
24.3.1.2.3;3.1.2.3. Mixing plate preparation;398
24.3.1.2.4;3.1.2.4. Assay plate preparation and data acquisition;398
24.3.1.2.5;3.1.2.5. Data analysis;399
24.3.2;3.2. 2 Confirmation and characterization screening assays;399
24.3.2.1;3.2.1. 5-TAMRA-RNA binding isotherms: Kd determination;399
24.3.2.2;3.2.2. Fluorescence anisotropy disruption assay: IC50 determination;399
24.3.2.3;3.2.3. Steady-state AP-labeled RNA fluorescence: Binding site localization and Kd determination;400
24.3.2.3.1;3.2.3.1. Data acquisition and analysis;400
24.3.2.3.2;3.2.3.2. Kd determination;401
24.3.2.4;3.2.4. Fluorescence-monitored thermal denaturation (Tm) assay;401
24.3.2.4.1;3.2.4.1. Reaction mixture preparation;401
24.3.2.4.2;3.2.4.2. Data acquisition and analysis;402
24.4;4. Conclusions;402
24.5;Acknowledgments;402
24.6;References;402
25;Chapter 19: Monitoring Ribosomal Frameshifting as a Platform to Screen Anti-Riboswitch Drug Candidates;406
25.1;1. Introduction;407
25.2;2. Materials;408
25.2.1;2.1. Nucleobases;408
25.2.2;2.2. Plasmid DNA template;408
25.2.3;2.3. In vitro transcription;410
25.2.4;2.4. Cell-free translation;410
25.2.5;2.5. Equipment;410
25.3;3. Methods;411
25.3.1;3.1. Preparation of nucleobases stock;411
25.3.2;3.2. Preparation of DNA template for in vitro transcription;411
25.3.3;3.3. In vitro transcription;411
25.3.4;3.4. Cell-free translation;412
25.3.5;3.5. Monitoring -1 FS;412
25.4;4. Notes;413
25.5;References;414
26;Author Index;416
27;Subject Index;434
28;Color Plate;445

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 of them cut across thematic boundaries by weaving together methodological solutions to multiple issues, 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 interval between discoveries of new riboswitch families widens. The series begins with two chapters outlining new methods that utilize informatics approaches in combination either with RNASeq and genome-wide methods (the Martin-Verstraete) or with in vitro selection (Lupták) 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 (Legault). 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 (Höbarter). 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 (Ferré-D’Amaré). 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 (Sintim) and large quantities of selectively 13C/15N-labeled RNA in previously unattainable labeling patterns for improved spectroscopic analysis (Dayie). 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 widely utilized SHAPE method of structural probing, along with details of how to implement new software for data interpretation (Weeks). 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 (Bevilacqua). The next chapter provides methods for obtaining appropriate crystals for ligand–RNA complexes, with emphasis on fragment-bound TPP riboswitches (Ferré-D’Amaré). 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 (Rambo). 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 (Greenbaum) and then with spin-label probes for electron paramagnetic resonance spectroscopy of large RNAs (Fanucci). 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 (Penedo, Walter, and Cornish). 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 (Smolke and Sigel). Finally, an innovative and relatively new technique known as DRaCALA is described in the last chapter of the first volume (Lee). 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 (Mörl). A contrasting approach uses in vitro selection/evolution to obtain ligand-responsive ligase ribozymes from highly diverse starting populations (Joyce), or to reshape and reprogram the ligand-binding and expression platforms of natural riboswitches (Batey). The next chapter presents methods for optimizing signal transduction (Kelley-Loughnane), since regulation sometimes benefits from maximizing suppression of basal expression in the OFF state and sometimes from maximizing expression in the ON state. 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” (Yomo), 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 (Jaffrey). The next two chapters describe how to use these modules in bacterial cells, first as intracellular sensors of intracellular cyclic dinucleotide levels (Hammond) and then for simultaneous and independent monitoring of mRNA and protein levels (Ellis). The next chapter takes this same question into solution and into vesicle-based artificial cells (Danelon). 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 (Ellington). 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 (Seeliger), as well as in more...