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Riboswitches as Targets and Tools

1st Edition - January 12, 2015

Editor: Donald H. Burke-Aguero

Hardback ISBN:

9 7 8 - 0 - 1 2 - 8 0 1 1 2 3 - 2

eBook ISBN:

9 7 8 - 0 - 1 2 - 8 0 1 3 3 6 - 6

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… Read more

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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.

Preface
- Volume 1
- Volume 2
Chapter One: Design of Transcription Regulating Riboswitches
- Abstract
- 1 Introduction
- 2 Computational Design of RNA Structures
- 3 Experimental Evaluation of Designed RNA Structures
- 4 Concluding Remarks
Chapter Two: Ligand-Dependent Exponential Amplification of Self-Replicating RNA Enzymes
- Abstract
- 1 Introduction
- 2 Exponential Amplification of RNA Enzymes
- 3 Ligand-Dependent Exponential Amplification
- 4 Nuclease-Resistant Autocatalytic Aptazymes
- 5 Real-Time Fluorescence Assays
- 6 Conclusions
- Acknowledgments
Chapter Three: Design of Modular “Plug-and-Play” Expression Platforms Derived from Natural Riboswitches for Engineering Novel Genetically Encodable RNA Regulatory Devices
- Abstract
- 1 Introduction
- 2 Design of Riboswitch Modules
- 3 Analysis of Riboswitch Activity Using an In Vitro Single-Turnover Transcription Assay
- 4 Cell-Based GFP Reporter Assay
- 5 Concluding Remarks
- Acknowledgment
Chapter Four: Integrating and Amplifying Signal from Riboswitch Biosensors
- Abstract
- 1 Introduction
- 2 Riboswitch Signal Integration
- 3 Riboswitch Signal Amplification Using Biological Circuitry
- Acknowledgments
Chapter Five: Simple Identification of Two Causes of Noise in an Aptazyme System by Monitoring Cell-Free Transcription
- Abstract
- 1 Theory
- 2 Equipment
- 3 Materials
- 4 Solutions and Buffers
- 5 Protocol
- 6 Step 1: Cell-Free Transcription–Translation and Fluorescence Monitoring
- 7 Step 2: Data Analysis
- 8 Step 3: (Optional) Quantification of the Intermediate RNAs
Chapter Six: Engineering of Ribosomal Shunt-Modulating Eukaryotic ON Riboswitches by Using a Cell-Free Translation System
- Abstract
- 1 Introduction
- 2 A Eukaryotic Translation Mechanism Requiring a Rigid mRNA Structure for Ribosomal Progression
- 3 Choice of a Translation System for Engineering Artificial Riboswitches
- 4 In Vitro-Selected Aptamer for Ribosomal Shunt-Modulating Riboswitches
- 5 How to Implant the Selected Aptamer into mRNA
- 6 General Design of mRNAs with Ribosomal Shunt-Modulating Riboswitches
- 7 Experiments
- 8 Conclusion
- Acknowledgments
Chapter Seven: Live-Cell Imaging of Mammalian RNAs with Spinach2
- Abstract
- 1 Introduction
- 2 Developing Spinach, an RNA Mimic of GFP
- 3 Imaging with Spinach2, a Superfolding Variant of Spinach
- 4 Fluorescence Imaging of Spinach2-Tagged RNAs
- 5 Imaging Other RNAs Using Spinach2
- Acknowledgments
Chapter Eight: In Vitro Analysis of Riboswitch–Spinach Aptamer Fusions as Metabolite-Sensing Fluorescent Biosensors
- Abstract
- 1 Introduction
- 2 Design and Preparation of an RNA-Based Fluorescent Biosensor
- 3 Determination of Ligand Selectivity and Affinity of Biosensor by Fluorescence Activation
- 4 Determination of Binding Kinetics of Biosensor
Chapter Nine: Using Spinach Aptamer to Correlate mRNA and Protein Levels in Escherichia coli
- Abstract
- 1 Introduction
- 2 Parts Selection and Plasmid Construction
- 3 E. coli Strain Selection
- 4 Culturing and Inducing E. coli Cells
- 5 Correlating mRNA and Protein Production Using Flow Cytometry
- 6 Correlating mRNA and Protein Production Using Fluorescence Microscopy
- 7 Summary
- Acknowledgments
Chapter Ten: Monitoring mRNA and Protein Levels in Bulk and in Model Vesicle-Based Artificial Cells
- Abstract
- 1 Introduction
- 2 The “Spinach Technology” for Combined Detection of mRNA and Protein in Cell-Free Expression Systems
- 3 Quantifying the Levels of mRNA and Protein Synthesized in PURE System Bulk Reactions
- 4 Detecting Gene Expression Inside Semipermeable Liposomes
- 5 Conclusion and Outlook
- Acknowledgments
Chapter Eleven: Design, Synthesis, and Application of Spinach Molecular Beacons Triggered by Strand Displacement
- Abstract
- 1 Introduction
- 2 How to Engineer Spinach Molecular Beacons Triggered by Toehold-Mediated Strand Displacement
- 3 How to Synthesize Spinach.ST Molecular Beacons Enzymatically
- 4 How to Perform Functional Assays of Spinach.ST Molecular Beacons
- 5 Application: Real-Time Spinach.ST-Based Detection of NASBA
- 6 Conclusions
- Acknowledgments
Chapter Twelve: Using Riboswitches to Regulate Gene Expression and Define Gene Function in Mycobacteria
- Abstract
- 1 Introduction
- 2 Riboswitch Reporter Assays
- 3 Construction of Recombinant Strains with Riboswitch-Regulated Genes
- 4 Induction of Mycobacterial Genes in Infected Host Cells
- Acknowledgments
Chapter Thirteen: Controlling Expression of Genes in the Unicellular Alga Chlamydomonas reinhardtii with a Vitamin-Repressible Riboswitch
- Abstract
- 1 Introduction
- 2 Design of the Repressible Riboswitch System Acting on Chloroplast Genes
- 3 Methods
- 4 Conclusions and Perspectives
- Acknowledgments
Chapter Fourteen: Conditional Control of Gene Expression by Synthetic Riboswitches in Streptomyces coelicolor
- Abstract
- 1 Introduction
- 2 Construction of Riboswitch-Controlled Expression Systems
- 3 Measurement of Riboswitch Activity
- 4 Characterization of Riboswitch-Controlled Gene Expression
- 5 Conclusion
- Acknowledgments
Chapter Fifteen: Engineering of Ribozyme-Based Aminoglycoside Switches of Gene Expression by In Vivo Genetic Selection in Saccharomyces cerevisiae
- Abstract
- 1 Theory
- 2 Equipment and Material
- 3 Protocol
Chapter Sixteen: Kinetic Folding Design of Aptazyme-Regulated Expression Devices as Riboswitches for Metabolic Engineering
- Abstract
- 1 Introduction
- 2 In Vitro Characterization
- 3 In Silico Transcript Design
- 4 In Vivo Validation
- 5 Future Directions
- Acknowledgment
Chapter Seventeen: Riboselector: Riboswitch-Based Synthetic Selection Device to Expedite Evolution of Metabolite-Producing Microorganisms
- Abstract
- 1 Introduction
- 2 Materials
- 3 Construction and Validation of Riboselector: Riboswitch-Based Synthetic Selection Devices
- 4 Application of Riboselector for Pathway Engineering
- 5 Concluding Remarks
- Acknowledgments
Chapter Eighteen: Fluorescence Assays for Monitoring RNA–Ligand Interactions and Riboswitch-Targeted Drug Discovery Screening
- Abstract
- 1 Introduction
- 2 General Considerations
- 3 Example Protocols
- 4 Conclusions
- Acknowledgments
Chapter Nineteen: Monitoring Ribosomal Frameshifting as a Platform to Screen Anti-Riboswitch Drug Candidates
- Abstract
- 1 Introduction
- 2 Materials
- 3 Methods
- 4 Notes
Author Index
Subject Index