Riboswitches as Targets and Tools (eBook)

Donald H. Burke-Aguero (Herausgeber)

eBook Download: PDF | EPUB

2015 | 1. Auflage
444 Seiten
Elsevier Science (Verlag)
978-0-12-801336-6 (ISBN)

Infos

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

Inhalt

Front Cover 1
Riboswitches as Targets and Tools 4
Copyright 5
Contents 6
Contributors 12
Preface 16
Volume 1 16
Volume 2 18
Chapter 1: Design of Transcription Regulating Riboswitches 22
1. Introduction 23
2. Computational Design of RNA Structures 26
2.1. The inverse folding problem 26
2.2. Designing multi-stable RNAs 30
2.3. Modeling external triggers 31
2.4. Current limitation of design software 33
3. Experimental Evaluation of Designed RNA Structures 36
3.1. Considerations on candidate selection and cloning procedures 36
3.2. Further characterization and current limitations 38
4. Concluding Remarks 39
References 40
Chapter 2: Ligand-Dependent Exponential Amplification of Self-Replicating RNA Enzymes 44
1. Introduction 45
2. Exponential Amplification of RNA Enzymes 46
2.1. Materials 47
2.2. Procedure for RNA self-replication 48
3. Ligand-Dependent Exponential Amplification 49
3.1. Procedure for quantitative ligand detection 50
3.2. Multiplexed ligand detection 51
3.3. Coupling ligand recognition to ligand-independent amplification 52
3.4. Procedure for coupled amplification 53
4. Nuclease-Resistant Autocatalytic Aptazymes 56
5. Real-Time Fluorescence Assays 57
6. Conclusions 59
Acknowledgments 59
References 59
Chapter 3: Design of Modular ``Plug-and-Play´´ Expression Platforms Derived from Natural Riboswitches for Engineering Nov... 62
1. Introduction 63
2. Design of Riboswitch Modules 67
2.1. Design strategy 67
2.2. Design optimization 75
3. Analysis of Riboswitch Activity Using an In Vitro Single-Turnover Transcription Assay 77
3.1. Template construction 77
3.1.1. Overlapping extension PCR 79
3.1.2. Purification of the template 80
3.2. Single-turnover in vitro transcription assay 81
4. Cell-Based GFP Reporter Assay 83
4.1. Reporter design 84
4.2. Protocol 84
4.3. Other considerations using in vivo reporters 87
5. Concluding Remarks 88
Acknowledgment 89
References 89
Chapter 4: Integrating and Amplifying Signal from Riboswitch Biosensors 94
1. Introduction 95
1.1. Biological circuits 95
2. Riboswitch Signal Integration 97
2.1. Design 97
2.1.1. AND gate 97
2.1.2. Selection of riboswitches 98
2.1.3. Using plasmid backbones available from the Registry of Standard Biological Parts 100
2.2. Build 100
2.2.1. Materials for construction of a riboswitch-based AND logic gate 100
2.2.2. Methods to construct pANDrs 102
2.3. Test 102
3. Riboswitch Signal Amplification Using Biological Circuitry 103
3.1. Design 103
3.2. Build 105
3.2.1. Materials for the construction of an amplification circuit 105
3.2.2. Methods to construct an amplification circuit, pAMPv1 106
3.2.2.1. PLas_RBS34(strong)_GFPa1_Terminator_Weak RBS_LasI +/-degradation tag 106
3.3. Test 106
3.4. Redesign and build 106
3.4.1. Methods to construct amplification circuit version 2 109
3.4.1.1. pAMPv2.1: PLas_RBS 34 (strong)_GFPa1_Terminator_RBS 33 (very weak)_RhlILVA 109
3.4.1.2. pAMPv2.2: PRhl_RBS 34 (strong)_GFPa1_Terminator_RBS 33 (very weak)_LasILVA 109
3.5. Test 109
3.5.1. Fluorescence activation 109
3.5.2. Signal progression 109
Acknowledgments 110
References 110
Chapter 5: Simple Identification of Two Causes of Noise in an Aptazyme System by Monitoring Cell-Free Transcription 114
1. Theory 115
2. Equipment 117
3. Materials 117
4. Solutions and Buffers 118
5. Protocol 119
5.1. Duration 119
5.2. Preparation 119
5.3. Caution 120
6. Step 1: Cell-Free Transcription-Translation and Fluorescence Monitoring 120
6.1. Overview 120
6.2. Duration 120
6.3. Tip 121
6.4. Tip 122
7. Step 2: Data Analysis 122
7.1. Overview 122
7.2. Duration 123
7.3. Tip 124
7.4. Tip 124
8. Step 3: (Optional) Quantification of the Intermediate RNAs 125
8.1. Overview 125
8.2. Duration 125
8.3. Tip 127
8.4. Tip 128
References 128
Chapter 6: Engineering of Ribosomal Shunt-Modulating Eukaryotic ON Riboswitches by Using a Cell-Free Translation System 130
1. Introduction 131
2. A Eukaryotic Translation Mechanism Requiring a Rigid mRNA Structure for Ribosomal Progression 133
3. Choice of a Translation System for Engineering Artificial Riboswitches 135
4. In Vitro-Selected Aptamer for Ribosomal Shunt-Modulating Riboswitches 136
5. How to Implant the Selected Aptamer into mRNA 137
6. General Design of mRNAs with Ribosomal Shunt-Modulating Riboswitches 140
6.1. 5 Untranslated region 140
6.2. Short open reading frame 140
6.3. Takeoff site 141
6.4. Aptamer-Ks conjugate 141
6.5. Landing site 141
6.6. Downstream open reading frame 141
6.7. 3 Untranslated region 142
7. Experiments 142
7.1. Construction of DNA templates for mRNAs 142
7.2. In vitro transcription 144
7.3. In vitro translation in WGE 144
7.4. Characterization of riboswitches 145
8. Conclusion 146
Acknowledgments 147
References 147
Chapter 7: Live-Cell Imaging of Mammalian RNAs with Spinach2 150
1. Introduction 150
2. Developing Spinach, an RNA Mimic of GFP 153
3. Imaging with Spinach2, a Superfolding Variant of Spinach 155
3.1. Tagging an RNA of interest with Spinach2 156
3.2. Testing a tagged RNA for Spinach2 fluorescence in vitro 156
3.3. Expressing 5S RNA in HEK 293-T cells 158
3.4. Expressing CGG60-Spinach2 in COS-7 cells 159
4. Fluorescence Imaging of Spinach2-Tagged RNAs 161
4.1. Imaging 5S-Spinach2 162
4.2. Imaging CGG60-Spinach2 163
5. Imaging Other RNAs Using Spinach2 165
Acknowledgments 165
References 165
Chapter 8: In Vitro Analysis of Riboswitch-Spinach Aptamer Fusions as Metabolite-Sensing Fluorescent Biosensors 168
1. Introduction 169
1.1. General equipment 171
1.2. General materials 171
2. Design and Preparation of an RNA-Based Fluorescent Biosensor 172
2.1. Preparation of DNA templates of riboswitch-Spinach aptamer fusion 173
2.1.1. Equipment and materials 175
2.1.2. Procedure for generating DNA template for transcription 175
2.2. Preparation of RNAs by in vitro transcription 175
2.2.1. Materials and equipment 175
2.2.2. Procedures 178
3. Determination of Ligand Selectivity and Affinity of Biosensor by Fluorescence Activation 181
3.1. Determination of ligand selectivity 181
3.1.1. Additional materials and equipment 181
3.1.2. Procedures 182
3.2. Determination of ligand affinity 185
4. Determination of Binding Kinetics of Biosensor 186
4.1. Determination of activation rate 188
4.2. Determination of deactivation rate 189
4.2.1. Additional materials and equipment 189
4.2.2. Procedure 190
References 192
Chapter 9: Using Spinach Aptamer to Correlate mRNA and Protein Levels in Escherichia coli 194
1. Introduction 195
2. Parts Selection and Plasmid Construction 196
3. E. coli Strain Selection 197
4. Culturing and Inducing E. coli Cells 199
5. Correlating mRNA and Protein Production Using Flow Cytometry 199
5.1. Prepping cells for analysis 200
5.2. Calibrating and setting up the flow cytometer 200
5.3. Analysis of flow data and presentation 201
6. Correlating mRNA and Protein Production Using Fluorescence Microscopy 201
6.1. Agarose pads 203
6.2. Prepping cells for analysis 203
6.3. Setting up the fluorescence microscope 204
6.4. Image analysis 205
7. Summary 205
Acknowledgments 206
References 206
Chapter 10: Monitoring mRNA and Protein Levels in Bulk and in Model Vesicle-Based Artificial Cells 208
1. Introduction 209
2. The ``Spinach Technology´´ for Combined Detection of mRNA and Protein in Cell-Free Expression Systems 211
2.1. Lighting up RNA with Spinach 211
2.2. Synthesis of DFHBI 212
2.3. Design and preparation of the DNA templates 213
2.4. In vitro transcription-translation with PUREfrex 214
2.5. Kinetics measurements by spectrofluorometry 215
2.6. Characterization of improved Spinach fluorescence in the PURE system 215
2.7. Orthogonal detection of synthesized mRNA-Spinach and protein 218
3. Quantifying the Levels of mRNA and Protein Synthesized in PURE System Bulk Reactions 219
3.1. Overall workflow 219
3.2. Preparation of reference RNA and purification 220
3.3. Gel analysis of mRNA concentration 220
3.4. Real-time quantitative PCR analysis 222
3.5. mRNA quantification from Spinach fluorescence of reference RNA 222
3.6. Calculating mYFP concentration by fluorescence correlation spectroscopy and absorbance measurements 223
3.6.1. Fluorescence correlation spectroscopy setup 223
3.6.2. Calibration of the detection volume 223
3.6.3. Measuring the concentration of synthesized mYFP 224
3.6.4. Converting mYFP fluorescence intensity into concentration 225
3.6.5. Absorbance measurements 226
3.6.6. FCS versus absorbance measurements 226
3.7. Quantitative analysis of mRNA and protein concentration versus time 226
4. Detecting Gene Expression Inside Semipermeable Liposomes 228
4.1. Preparation of lipid film-coated beads 229
4.2. Liposome formation and encapsulation of the biosynthesis machinery 229
4.3. Surface functionalization, liposome immobilization, and triggering of gene expression 230
4.4. Visualizing liposomes with fluorescence microscopy 231
4.5. Factors influencing the levels of mRNA and protein produced in liposomes 231
5. Conclusion and Outlook 232
Acknowledgments 233
References 233
Chapter 11: Design, Synthesis, and Application of Spinach Molecular Beacons Triggered by Strand Displacement 236
1. Introduction 237
2. How to Engineer Spinach Molecular Beacons Triggered by Toehold-Mediated Strand Displacement 239
2.1. Engineering conformations and sequence modules for Spinach beacons 239
2.2. Using NUPACK and KineFold for design 244
2.3. Promoters for Spinach.ST expression 246
2.4. Stabilization of Spinach.ST within a tRNA scaffold 247
2.5. Programming triggers 247
2.5.1. Designing triggers to discriminate single-nucleotide mismatches 248
2.5.2. Designing triggers to function in the context of nucleic acid circuits 248
2.5.3. Associative toehold triggers 251
2.6. Spinach.ST function within larger RNA contexts 252
3. How to Synthesize Spinach.ST Molecular Beacons Enzymatically 254
3.1. DNA oligonucleotides 254
3.2. How to generate transcription templates 254
4. How to Perform Functional Assays of Spinach.ST Molecular Beacons 255
4.1. In vitro transcription reactions 255
4.2. Reactions with added trigger and control sequences 256
4.3. Real-time cotranscriptional functional assays with Spinach.ST 257
5. Application: Real-Time Spinach.ST-Based Detection of NASBA 258
5.1. Including Spinach.ST trigger components in NASBA primers 258
5.2. Detecting NASBA amplification with Spinach.ST 264
6. Conclusions 265
Acknowledgments 266
References 266
Chapter 12: Using Riboswitches to Regulate Gene Expression and Define Gene Function in Mycobacteria 272
1. Introduction 273
2. Riboswitch Reporter Assays 274
2.1. Construction of promoter-riboswitch GFP or ß-galactosidase reporter plasmids 279
2.2. GFP fluorescence endpoint assay 280
2.2.1. Note on culturing bacteria in multi-well plates 281
2.3. GFP flow cytometry 281
2.4. ß-Galactosidase activity endpoint assay 282
3. Construction of Recombinant Strains with Riboswitch-Regulated Genes 283
4. Induction of Mycobacterial Genes in Infected Host Cells 284
Acknowledgments 285
References 285
Chapter 13: Controlling Expression of Genes in the Unicellular Alga Chlamydomonas reinhardtii with a Vitamin-Repressible ... 288
1. Introduction 289
2. Design of the Repressible Riboswitch System Acting on Chloroplast Genes 290
3. Methods 295
3.1. Growth conditions 295
3.2. Nuclear transformation 296
3.3. Chloroplast transformation 297
3.4. Screening for essential chloroplast genes 298
3.5. Effect of vitamins 299
4. Conclusions and Perspectives 299
Acknowledgments 300
References 300
Chapter 14: Conditional Control of Gene Expression by Synthetic Riboswitches in Streptomyces coelicolor 304
1. Introduction 305
2. Construction of Riboswitch-Controlled Expression Systems 307
2.1. Vector 307
2.2. Riboswitch design 308
2.3. Genetic manipulations in S. coelicolor 310
3. Measurement of Riboswitch Activity 310
3.1. ß-Glucuronidase measurement 310
3.2. Detection on agar plates 310
3.3. Measurement in liquid culture 311
4. Characterization of Riboswitch-Controlled Gene Expression 311
4.1. Dynamic range of regulation can be adjusted by appropriate promoter-riboswitch pairing 311
4.2. Dose dependence of riboswitch regulation 316
4.3. Assessing kinetics of induction and repression 317
5. Conclusion 317
Acknowledgments 318
References 319
Chapter 15: Engineering of Ribozyme-Based Aminoglycoside Switches of Gene Expression by In Vivo Genetic Selection in Sacc... 322
1. Theory 323
2. Equipment and Material 327
2.1. Molecular subcloning of the aptazyme library and propagation into Escherichia coli 328
2.2. Selection and hit identification of the aptazyme library 330
3. Protocol 331
3.1. Aptazyme library construction 331
3.2. Transformation of the aptazyme library into E. coli XL10 gold 335
3.3. Generation of electrocompetent yeast cells 336
3.4. Selection and screening of the yeast aptazyme library and ``hit´´ identification 337
References 339
Chapter 16: Kinetic Folding Design of Aptazyme-Regulated Expression Devices as Riboswitches for Metabolic Engineering 342
1. Introduction 343
2. In Vitro Characterization 345
2.1. Materials and equipment 347
2.2. Methods 348
2.2.1. DNA oligo purification 348
2.2.2. Ethanol precipitation 350
2.2.3. Cotranscriptional cleavage analysis 350
3. In Silico Transcript Design 352
3.1. System and submission guidelines 354
3.2. Computational methods 355
4. In Vivo Validation 357
4.1. Equipment and reagents 358
4.2. Methods 358
5. Future Directions 359
Acknowledgment 360
References 360
Chapter 17: Riboselector: Riboswitch-Based Synthetic Selection Device to Expedite Evolution of Metabolite-Producing Micro ... 362
1. Introduction 363
2. Materials 365
2.1. Equipment 365
2.2. Materials 365
2.3. Oligonucleotides 365
3. Construction and Validation of Riboselector: Riboswitch-Based Synthetic Selection Devices 365
3.1. Riboselector based on a natural riboswitch 365
3.1.1. Construction of a device for reporting its operation 370
3.1.2. Characterization of the constructed reporting device 371
3.2. Riboselector based on an artificial riboswitch from synthetic aptamer 372
3.2.1. Library generation for selection of synthetic device 372
3.2.2. Iterative positive and negative selection of synthetic device 373
3.2.3. Characterization of potential synthetic device 375
3.3. Characterization and validation of the constructed Riboselector 375
3.3.1. Growth rate characterization with Riboselector under selective pressure 375
3.3.2. Growth competition with Riboselector under selective pressure 376
4. Application of Riboselector for Pathway Engineering 376
4.1. Metabolic pathway engineering for phenotypic diversification 376
4.1.1. Pathway engineering to enhance lysine production 378
4.1.2. Library generation for optimization of ppc expression level 379
4.2. Pathway optimization using synthetic selection device 380
4.2.1. Setting an appropriate selection pressure by measuring growth rates 380
4.2.2. Enrichment experiment and analysis 380
5. Concluding Remarks 381
Acknowledgments 381
References 382
Chapter 18: Fluorescence Assays for Monitoring RNA-Ligand Interactions and Riboswitch-Targeted Drug Discovery Screening 384
1. Introduction 385
1.1. Noncoding RNAs and drug discovery 385
1.2. Screening cascade for RNA-targeted drug discovery 387
2. General Considerations 390
2.1. Equipment 390
2.2. Setup and optimization 390
2.2.1. Microplate setup and spectrometer settings 390
2.2.2. Ligand fluorescence 391
2.3. Pipetting and assay preparation 391
2.4. Materials and reagents 392
3. Example Protocols 394
3.1. 1 Screening assays 394
3.1.1. Steady-state fluorescence assay: Ligand-antiterminator RNA binding assay 394
3.1.1.1. Materials and reagents 394
3.1.1.2. Reagent plate preparation 395
3.1.1.3. Mixing plate preparation 395
3.1.1.4. Assay plate preparation and data acquisition 396
3.1.1.5. Analysis 396
3.1.2. Fluorescence anisotropy assay: Ligand-induced tRNA-antiterminator complex disruption assay 397
3.1.2.1. Materials and reagents 397
3.1.2.2. Reagent plate preparation 398
3.1.2.3. Mixing plate preparation 398
3.1.2.4. Assay plate preparation and data acquisition 398
3.1.2.5. Data analysis 399
3.2. 2 Confirmation and characterization screening assays 399
3.2.1. 5-TAMRA-RNA binding isotherms: Kd determination 399
3.2.2. Fluorescence anisotropy disruption assay: IC50 determination 399
3.2.3. Steady-state AP-labeled RNA fluorescence: Binding site localization and Kd determination 400
3.2.3.1. Data acquisition and analysis 400
3.2.3.2. Kd determination 401
3.2.4. Fluorescence-monitored thermal denaturation (Tm) assay 401
3.2.4.1. Reaction mixture preparation 401
3.2.4.2. Data acquisition and analysis 402
4. Conclusions 402
Acknowledgments 402
References 402
Chapter 19: Monitoring Ribosomal Frameshifting as a Platform to Screen Anti-Riboswitch Drug Candidates 406
1. Introduction 407
2. Materials 408
2.1. Nucleobases 408
2.2. Plasmid DNA template 408
2.3. In vitro transcription 410
2.4. Cell-free translation 410
2.5. Equipment 410
3. Methods 411
3.1. Preparation of nucleobases stock 411
3.2. Preparation of DNA template for in vitro transcription 411
3.3. In vitro transcription 411
3.4. Cell-free translation 412
3.5. Monitoring -1 FS 412
4. Notes 413
References 414
Author Index 416
Subject Index 434
Color Plate 445

Textauszug

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...

Produkt-Details

• Erscheint lt. Verlag: 12.1.2015
• Sprache: englisch
• Themenwelt: Medizin / Pharmazie ► Medizinische Fachgebiete
• Themenwelt: Naturwissenschaften ► Biologie ► Biochemie
• Themenwelt: Naturwissenschaften ► Biologie ► Genetik / Molekularbiologie
• Themenwelt: Naturwissenschaften ► Physik / Astronomie ► Angewandte Physik
• ISBN-10: 0-12-801336-2 / 0128013362
• ISBN-13: 978-0-12-801336-6 / 9780128013366