RNA Structure Determination: Methods and Protocols (Methods in Molecular Biology, 1490)

✍ Scribed by Douglas H. Turner (editor), David H. Mathews (editor)

• Publisher: Humana
• Year: 2016
• Tongue: English
• Leaves: 295
• Category: Library

✦ Synopsis

This volume provides protocols and procedures for determining and modeling RNA structure. Chapters guide the reader through protocols for RNA secondary structure prediction, single sequence modeling, Crumple, RNAstructure to model conserved secondary structures with multiple homologs, the prediction of bimolecular secondary structures with RNAstructure, STarMir, protocols for structure mapping, mapping data to constrain or restrain RNA secondary structure prediction with RNAstructure, unassigned NMR resonances, modeling protocols for Rosetta FARFAR, RNAComposer , ModeRNA, and MC-Fold. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Authoritative and Practical, RNA Structure Determination:Methods and Protocols aims to ensure successful results in the further study of this vital field.

✦ Table of Contents

Preface
Twenty-First Century Advances
Organization of the Book
Contents
Contributors
Chapter 1: Crumple: An Efficient Tool to Explore Thoroughly the RNA Folding Landscape
1 Introduction
2 Materials
3 Methods
3.1 Crumple Local Installation
3.1.1 Downloading and Extracting the Crumple Package
3.1.2 Installing GNU Make and Compiling Crumple
3.2 Using Crumple Features and Commands
3.2.1 Crumple Output Syntax
3.2.2 Crumple Input and Output
3.3 Running Parallelized Crumple
3.3.1 Materials and Compilation
3.3.2 Parallel Output Efficiency
4 Notes
4.1 Use with Sliding Windows and Assembly
4.2 Use with Thermodynamic Parameters
References
Chapter 2: Secondary Structure Prediction of Single Sequences Using RNAstructure
1 Introduction
1.1 Free Energy Minimization
1.2 Partition Functions
1.3 Pseudoknot Prediction
2 Protocols
2.1 Installing the Graphical User Interface
2.2 Sequence Input and Editing
2.3 Fold: Predict Minimum Free Energy Structure
2.4 Energy Dot Plot: Show Well-
2.5 Partition Function Calculation: Color Annotate Structure with Base Pair Probabilities
2.6 MaxExpect: Predict a Structure Composed of Probable Pairs
2.7 Stochastic Sampling: Sample a Set of Structures
2.8 ProbKnot: Predict Structures That May Contain Pseudoknots
2.9 Efn2: Calculate The Free Energy Change of a Given Structure
2.10 Text User Interface (TUI) (Command Line Interface)
2.11 SEQ and CT File Formats
2.12 DOT2CT and CT2DOT: Structure File Conversions
2.13 CircleCompare: To Visually Compare Two Structures
3 Notes
References
Chapter 3: Prediction of Secondary Structures Conserved in Multiple RNA Sequences
1 Introduction
2 Protocols
2.1 Dynalign: Predict Consensus Structures of a Pair of Sequences and Its Associated Alignment
2.2 Multilign: Predict Consensus Structures of Multiple Sequences and Associated Alignments
2.3 TurboFold: Predict Structures of Multiple Sequences
2.4 PARTS
3 Notes
References
Chapter 4: Predicting RNA–RNA Interactions Using RNAstructure
1 Introduction
1.1 Bifold
1.2 Bipartition
1.3 DuplexFold
1.4 AccessFold
2 Materials
3 Methods
3.1 Download and Install RNAstructure
3.2 Enter Sequences
3.3 Using Bifold with the GUI
3.4 Bipartition
3.5 DuplexFold
3.6 AccessFold
4 Notes
References
Chapter 5: A Method to Predict the Structure and Stability of RNA/RNA Complexes
1 Introduction
2 Tertiary Loop Entropy Calculation
3 Methods
3.1 RNA/RNA Complex Structure Prediction Based on Complete Structure Enumeration
3.2 Two-Step Screening Method for RNA/RNA Complex Prediction
4 Notes
References
Chapter 6: STarMir Tools for Prediction of microRNA Binding Sites
1 Introduction
1.1 Identification of miRNA Binding Sites
2 Materials
3 Methods
3.1 Web Protocol for Using STarMir
3.2 STarMir Input Page
3.2.1 Model
3.2.2 Species
3.2.3 miRNA
3.2.4 mRNA
3.2.5 mRNA Region
3.2.6 mRNA Name
3.2.7 Email Address
3.3 STarMir Output
4 Notes
References
Chapter 7: Traditional Chemical Mapping of RNA Structure In Vitro and In Vivo
1 Introduction
2 Material
2.1 Equipment
2.2 RNA Preparation
2.3 End Labeling of RNAs and of Oligo-deoxyribonucleotide
2.4 Modification Reaction
2.5 Fractionation of End-Labeled RNA Fragments
2.6 Detection of Cleavages by Primer Extension
2.7 Fractionation of Cleaved Fragments by Polyacrylamide–Urea Gel Electrophoresis
3 Methods
3.1 Establishing the Protocols
3.2 Choice of the Detection Method
3.3 RNA Preparation
3.4 Lead(II)-Induced Cleavages of RNAs In Vitro
3.5 Base-Specific Modification of RNA In Vitro
3.5.1 DMS Modification (N3C, N1A)
3.5.2 DMS Modification (N7G)
3.5.3 CMCT Modification (N3U, N1G)
3.5.4 DEPC Carbethoxylation (N7A)
3.5.5 Kethoxal Modification (N1,N2G)
3.6 Fractionation of End-Labeled RNA Fragments
3.6.1 Ladders for Cleavage Assignments
3.6.2 Purification of End-Labeled RNA Fragments by PAGE
3.7 Detection of Cleavages or Modifications by Primer Extension
3.7.1 Hybridization and Primer Extension
3.7.2 Gel Fractionation of Labeled cDNA Fragments
3.8 In Vivo Lead(II)-Induced Cleavages
3.9 In Vivo DMS Modification
4 Notes
References
Chapter 8: High-Throughput Nuclease Probing of RNA Structures Using FragSeq
1 Introduction
1.1 Considerations in Designing a FragSeq Experiment
2 Materials
2.1 Purification of Complex RNA Mixture for Probing
2.2 Nuclease Calibration and Digestion of RNA with Nuclease P1
2.3 Bioinformatics Analysis
3 Methods
3.1 Calibration of P1 Nuclease for Probing a Complex RNA Mixture
3.1.1 Calibration Experimental Workflow
3.1.2 Choosing a Nuclease Condition
3.2 Digestion of a Complex RNA Mixture with Nuclease P1
3.3 Ligation and Library Prep from Nuclease and Control RNA Samples
3.4 Summary of Steps in the Bioinformatics Analysis Pipeline
3.5 Mapping Sequencing Reads to Reference Sequence
3.5.1 Special Considerations for Spliced or Overlapping RNAs
3.5.2 Special Considerations for Multi-copy or Repetitive RNAs
3.6 Running the  FragSeq Command-Line Tool (reads ToStruct.py)
3.6.1 Overview
3.6.2 Input Files
3.6.3 Configuration Files
3.7 Working with the Output of reads ToStruct. py
3.7.1 Interpretation of Cutting Scores
3.7.2 Genome Browser Output
3.7.3 Output for RNA Structure Analysis
4 Notes
References
Chapter 9: Mapping RNA Structure In Vitro with SHAPE Chemistry and Next-Generation Sequencing (SHAPE-Seq)
1 Introduction
2 Materials
2.1 RNA Modification Components
2.2 RNA Ligation Components
2.3 Reverse Transcription (RT) Components
2.4 ssDNA Ligation and Quality Analysis (QA) Components
2.5 Library Construction Components
2.6 NGS and Data Analysis
3 Methods
3.1 RNA Modification
3.2 RNA Ligation
3.3 Reverse Transcription
3.4 ssDNA Ligation and Quality Analysis
3.5 Library Construction
3.6 NGS and Data Analysis
4 Notes
References
Chapter 10: Experiment-Assisted Secondary Structure Prediction with RNAstructure
1 Introduction
2 Protocols
2.1 RNAstructure Graphical User Interface for Experimental Data
2.2 Incorporating Traditional Chemical and Enzymatic Constraints into Secondary Structure Prediction
2.3 Incorporating SHAPE Data into Secondary Structure Prediction
2.4 File Formats
2.5 Alternative Protocols: Text User Interface
3 Notes
References
Chapter 11: RNA Secondary Structure Determination by NMR
1 Introduction
1.1 NAPSS-CS Calculation
2 Materials
3 Methods
3.1 Protocol for Compiling Program
3.2 Creating Sequence and Constraint Files
3.3 Running NAPSS-CS
4 Notes
References
Chapter 12: Modeling Small Noncanonical RNA Motifs with the Rosetta FARFAR Server
1 Introduction
1.1 FARFAR Calculation
2 Materials
3 Methods
3.1 Main Page Form
3.2 Advanced Options
3.3 Server Results
References
Chapter 13: Automated RNA 3D Structure Prediction with RNAComposer
1 Introduction
1.1 RNA 3D Structure Prediction with RNAComposer
2 Materials
3 Methods
3.1 RNAComposer Website at a Glance
3.2 Getting Started: How to Prepare the Input Data
3.3 Structure Modeling in the Interactive Mode
3.4 Creating User Account
3.5 Structure Modeling in the Batch Mode
3.5.1 Atom Distance Restraints
3.5.2 RNA 3D Structure Prediction Results
3.6 Application Example
3.7 User Workspace
4 Notes
References
Chapter 14: RNA 3D Structure Modeling by Combination of Template-­Based Method ModeRNA, Template-Free Folding with SimRNA, and Refinement with QRNAS
1 Introduction
2 Materials
3 Methods
3.1 Template Search
3.2 Preparation of the Template Structure for Modeling
3.3 Target-Template Alignment
3.4 Building a Model with ModeRNA
3.5 Template-Free Refolding of Sequence Fragments with SimRNA
3.6 RNA 3D Structure Refinement
3.7 RNA 3D Structure Quality Evaluation
References
Chapter 15: Exploring Alternative RNA Structure Sets Using MC-Flashfold and db2cm
1 Introduction
1.1 Secondary Structure Representations
2 Materials
3 Methods
3.1 Installing the Software
3.1.1 Obtaining the Software
3.1.2 Understanding the Software Package
MC-Flashfold
FlashScan
db2cm
3.1.3 Compiling db2cm
3.2 Folding an RNA Sequence Using MC-Flashfold
3.3 Creating Dot Plots and Arc Plots Using db2cm
3.4 Viewing the Plots Using Chrome
3.5 Tailoring the Plots
3.5.1 Adjusting the Contrast
3.5.2 Choosing the Threshold
3.5.3 Controlling Which Elements Get Plotted
3.5.4 Customizing Image Size, Rotation and Zoom Level
3.5.5 Editing the Plots in Adobe Illustrator
3.6 Viewing the trp-­operon Leader Anti-terminator and Terminator Stem-Loops
3.6.1 Folding the Sequences
3.6.2 Generating the Plots
3.6.3 Drawing the Full Plots for Different Numbers of Structures
3.6.4 Revealing the Anti-terminator Loop in the Full-­Length trp-leader
3.6.5 Simulating Transcription Elongation
4 Notes
4.1 Updating Your Software
4.2 Fixing Copy and Paste of Commands
4.3 Exploring Command Line Tools
4.4 Using Folding Masks
4.5 Forgetting the –v Parameter
References
Chapter 16: NMR Methods for Characterization of RNA Secondary Structure
1 Introduction
2 Experimental Considerations
3 Revealing Canonically Base Paired Stems
4 Data Acquisition
5 Secondary Structure Prediction
6 3D Structure Determination
References
Chapter 17: The Quick and the Dead: A Guide to Fast Phasing of Small Ribozyme and Riboswitch Crystal Structures
1 Introduction
1.1 Rationale for the Choice of Iridium and Osmium Amines for RNA Phasing
1.2 Observed Metal Binding Sites in Case Studies
2 Materials
3 Methods
3.1 Finding Anomalous Signal-to-Noise Ratios as a Function of Resolution
3.2 Locating Heavy Atom Sites with the ShelxD Program (Called XM by Bruker)
3.3 Phase Calculations and Noise Suppression Using the ShelxE Program
3.4 Options for Converting Files Shelx (.hkl) or Scalepack (.sca) Format to .mtz Format
3.5 Using Resolve for Further Density Modification and Phenix/Resolve for Optional RNA Autobuilding
4 Notes
References
Index