Riboswitch Discovery, Structure and Function
- 1st Edition, Volume 549 - November 26, 2014
- Latest edition
- Editor: Donald H. Burke-Aguero
- Language: English
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 method… Read more
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
Biochemists, biophysicists, molecular biologists, analytical chemists, and physiologists.
- Preface
- Volume 1
- Volume 2
- Part I: Riboswitch Discovery
- Chapter One: Riboswitch Discovery by Combining RNA-Seq and Genome-Wide Identification of Transcriptional Start Sites
- Abstract
- 1 Introduction
- 2 RNA Isolation and mRNA Enrichment
- 3 Genome-Wide Mapping of Transcription Start Sites by dRNA-Seq
- 4 Genome-Wide Analysis of Transcript Length by RNA-Seq
- 5 Processing and Analysis of dRNA-Seq and RNA-Seq Data
- 6 Characterization of New Potential Riboswitches Using dRNA-Seq and RNA-Seq Analyses
- Chapter Two: Discovering Human RNA Aptamers by Structure-Based Bioinformatics and Genome-Based In Vitro Selection
- Abstract
- 1 Introduction
- 2 Precautions
- 3 Generating a Human Genomic DNA Pool
- 4 In Vitro Selection of RNA Aptamers
- 5 Structure-Based Searches for Naturally Occurring Aptamers
- Chapter One: Riboswitch Discovery by Combining RNA-Seq and Genome-Wide Identification of Transcriptional Start Sites
- Part II: Synthesis and Sample Prep Methods for Large RNAs
- Chapter Three: Affinity Purification of In Vitro Transcribed RNA with Homogeneous Ends Using a 3′-ARiBo Tag
- Abstract
- 1 Introduction
- 2 Batch Affinity Purification of RNA Using a 3′-ARiBo Tag
- 3 Ensuring 5′-Homogeneity of Affinity-Purified RNA
- 4 Summary
- Acknowledgments
- Chapter Four: Deoxyribozyme-Mediated Ligation for Incorporating EPR Spin Labels and Reporter Groups into RNA
- Abstract
- 1 Introduction
- 2 Synthesis of Spin-Labeled RNA Using Convertible Nucleosides
- 3 DNA-Catalyzed Ligation of RNA Using 9DB1*
- 4 Protocols for Synthesis of Spin-Labeled SAM-I Riboswitch
- 5 General Considerations and Future Developments
- Acknowledgments
- Chapter Five: A Flexible, Scalable Method for Preparation of Homogeneous Aminoacylated tRNAs
- Abstract
- 1 Introduction
- 2 Methods
- Acknowledgments
- Chapter Six: Synthesis of a Biotinylated Photocleavable Nucleotide Monophosphate for the Preparation of Natively Folded RNAs
- Abstract
- 1 Theory
- 2 Equipment
- 3 Materials
- 4 Protocol
- 5 Step 1: Synthesis of Biotin-PC GMP
- 6 Step 2: Transcription of D5 and Ribosomal A-site RNAs Using Unmodified GTP and Biotin-PC GMP
- 7 Step 3: Purification of Biotin-Labeled RNA with Affinity Avidin Column and Photocleavage
- 8 Conclusions
- Acknowledgments
- Chapter Seven: Chemo-Enzymatic Synthesis of Selectively 13C/15N-Labeled RNA for NMR Structural and Dynamics Studies
- Abstract
- 1 Theory
- 2 Equipment
- 3 Materials
- 4 Protocol
- 5 Step 1: Synthesis of Uracil
- 6 Step 2: Synthesis of UTP
- 7 Step 3: Synthesis of CTP
- 8 Step 4: Purification and Quantification
- 9 Step 5: Quality Control
- 10 Step 6: In Vitro RNA Transcription
- 11 Step 7: NMR Applications
- 12 Conclusion
- Acknowledgments
- Chapter Three: Affinity Purification of In Vitro Transcribed RNA with Homogeneous Ends Using a 3′-ARiBo Tag
- Part III: Structure and Folding
- Chapter Eight: SHAPE Analysis of Small RNAs and Riboswitches
- Abstract
- 1 Theory
- 2 Equipment
- 3 Materials
- 4 Protocol
- 5 Step 1: RNA Folding and SHAPE Probing
- 6 Step 2: Primer Extension
- 7 Step 3: Capillary Electrophoresis
- 8 Step 4: Data Processing Using QuShape
- 9 Step 5: Data Processing and RNA Modeling
- Acknowledgments
- Chapter Nine: Experimental Approaches for Measuring pKa’s in RNA and DNA
- Abstract
- 1 Introduction
- 2 Experimental Parameters for pH Titrations
- 3 RNA Cleavage Kinetics
- 4 Spectroscopic-Detected Methods
- 5 Perspective
- Acknowledgments
- Chapter Ten: Crystallographic Analysis of TPP Riboswitch Binding by Small-Molecule Ligands Discovered Through Fragment-Based Drug Discovery Approaches
- Abstract
- 1 Introduction
- 2 Methods
- 3 Conclusions
- Acknowledgments
- Chapter Eleven: Methods for Using New Conceptual Tools and Parameters to Assess RNA Structure by Small-Angle X-Ray Scattering
- Abstract
- 1 Introduction
- 2 Specialized Equipment
- 3 Preparation of the RNA for a SAXS Study
- 4 Interpretation of the X-Ray Scattering Curve
- 5 Case Studies
- 6 Multiphase Volumetric Modeling
- 7 Gold Labels and Comprehensive Conformations
- 8 Considerations
- Acknowledgments
- Chapter Eight: SHAPE Analysis of Small RNAs and Riboswitches
- Part IV: Dynamics
- Chapter Twelve: Use of 19F NMR Methods to Probe Conformational Heterogeneity and Dynamics of Exchange in Functional RNA Molecules
- Abstract
- 1 Introduction
- 2 Methods
- 3 Conclusion and Remarks
- Acknowledgments
- Chapter Thirteen: Site-Directed Spin-Labeling Strategies and Electron Paramagnetic Resonance Spectroscopy for Large Riboswitches
- Abstract
- 1 Techniques Used for Riboswitch Studies
- 2 Ligation Methods for SDSL of Large Riboswitches
- 3 CW-EPR Spectral Analysis of Riboswitches
- Chapter Fourteen: Using sm-FRET and Denaturants to Reveal Folding Landscapes
- Abstract
- 1 Introduction
- 2 Single-Molecule FRET: Technical Aspects
- 3 Riboswitch Structure and Biological Function
- 4 Combination of sm-FRET and Denaturants to Investigate Riboswitch Folding
- 5 Summary and Prospects
- Chapter Fifteen: Riboswitch Structure and Dynamics by smFRET Microscopy
- Abstract
- 1 Introduction
- 2 Methods
- 3 Practical Experimental Considerations
- 4 Data Analysis
- 5 Induced-Fit Versus Conformational Selection
- 6 Summary and Conclusions
- Acknowledgments
- Chapter Sixteen: Ribosome Structure and Dynamics by smFRET Microscopy
- Abstract
- 1 Introduction
- 2 Overview of Ribosome Structure and Function
- 3 Methodology
- 4 Ribosome Dynamics
- 5 Data Acquisition
- 6 Building and Verifying Histograms, Normalization, Gaussian Fitting
- 7 Future Directions
- Chapter Seventeen: Unraveling the Thermodynamics and Kinetics of RNA Assembly: Surface Plasmon Resonance, Isothermal Titration Calorimetry, and Circular Dichroism
- Abstract
- 1 The RNA Folding Problem and Assembly of RNA Tertiary Structure
- 2 Practical Aspects of Biophysical Studies of RNA Assembly
- 3 Specific Measurements of Ion-Driven RNA Assembly
- 4 Concluding Remarks
- Acknowledgments
- Chapter Twelve: Use of 19F NMR Methods to Probe Conformational Heterogeneity and Dynamics of Exchange in Functional RNA Molecules
- Part V: Ligand Interactions
- Chapter Eighteen: ITC Analysis of Ligand Binding to PreQ1 Riboswitches
- Abstract
- 1 Introduction
- 2 Experimental Procedures for ITC
- Acknowledgments
- Chapter Nineteen: Facile Characterization of Aptamer Kinetic and Equilibrium Binding Properties Using Surface Plasmon Resonance
- Abstract
- 1 Introduction
- 2 Materials
- 3 Sensor Surface Immobilization
- 4 Characterization of Aptamer Binding Properties
- 5 Conclusion
- Acknowledgments
- Chapter Twenty: The AdoCbl–Riboswitch Interaction Investigated by In-Line Probing and Surface Plasmon Resonance Spectroscopy (SPR)
- Abstract
- 1 Introduction
- 2 In-Line Probing Experiments
- 3 SPR Spectroscopy
- 4 Conclusion
- Acknowledgments
- Chapter Twenty-One: Assessing RNA Interactions with Proteins by DRaCALA
- Abstract
- 1 Introduction
- 2 DRaCALA-Based Detection of Protein–Ligand Interactions
- 3 Principle of DRaCALA
- 4 Determination of Fraction Bound by DRaCALA
- 5 Steps for Performing DRaCALA to Detect Protein Interaction With RNA
- 6 CsrA Binds Specifically to RsmY and RsmZ
- 7 Other Modifications of DRaCALA for RNA–Protein Interactions
- Chapter Eighteen: ITC Analysis of Ligand Binding to PreQ1 Riboswitches
- Author Index
- Subject Index
- Edition: 1
- Latest edition
- Volume: 549
- Published: November 26, 2014
- Language: English
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Donald H. Burke-Aguero
Affiliations and expertise
Department of Molecular Microbiology & Immunology and Department of Biochemistry, University of Missouri, USARead Riboswitch Discovery, Structure and Function on ScienceDirect