RNA Structure and Dynamics (Methods in Molecular Biology, 2568)

Description

This volume provides a wide spectrum of multidisciplinary approaches for studying RNA structure and dynamics, including detailed accounts of experimental and computational procedures. Chapters guide readers through cryo-electron microscopy, crystallography, isothermal titration calorimetry, small angle X-ray scattering, single-molecule Förster Energy transfer, X-ray free electron laser,  atomic force microscopy, computational simulation, and prediction.  Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and reagents, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols. Authoritative and cutting-edge, RNA Structure and Dynamics aims to be a foundation for future studies and to be a source of inspiration for new investigations in the field.   Preface Contents Contributors Chapter 1: Cotranscriptional Assembly and Native Purification of Large RNA-RNA Complexes for Structural Analyses 1 Introduction 2 Materials 3 Methods 3.1 Choice of T7 Promoter and Desired RNA 5′ Phosphorylation State 3.2 (Optional) Homogenization of RNA Termini by Dual Ribozyme Processing 3.3 Streamlined DNA Template Preparation and Cotranscriptional Assembly of RNA-RNA Complexes 3.4 Purification of Large RNA-RNA Complexes by Size-Exclusion Chromatography (SEC) 3.5 Purification of Large RNA-RNA Complexes by Anion Exchange Chromatography 4 Notes References Chapter 2: Probing RNA Structures and Interactions Using Fluorescence Lifetime Analyses 1 Introduction 2 Materials 3 Methods 3.1 Semi-enzymatic Synthesis of 2AP-Labeled tRNA 3.2 Instrument Setup and Validation 3.3 Benchmarking Fluorescence Lifetimes 3.4 Measure Fluorescence Lifetimes of the Experimental Systems Under Study 4 Notes References Chapter 3: Characterizing Fluorescence Properties of Turn-on RNA Aptamers 1 Introduction 2 Materials 2.1 In Vitro Transcription and RNA Purification 2.2 Steady-State Fluorescence Measurements 2.3 Fluorescence Lifetime Measurements 3 Methods 3.1 In Vitro Transcription 3.2 Steady-State Fluorescence 3.2.1 Binding Affinity Measurement by Steady-State Fluorescence 3.2.2 Fluorescence Enhancement Measurement by Steady-State Fluorescence 3.3 Fluorescence Lifetime Measurement 3.4 Fluorescence Lifetime Analysis 4 Notes References Chapter 4: Probing Transient Riboswitch Structures via Single Molecule Accessibility Analysis 1 Introduction 2 Materials 2.1 Ligand 7-aminomethyl-7-deazaguanine (preQ1) 2.2 Riboswitch Containing mRNA Transcript 2.3 Biotinylated Bovine Serum Albumin (1 mg/ml) 2.4 T50 Buffer 2.5 Oxygen Scavenging Stock Solution with TROLOX (OSST) 2.6 2x Reconstitution Buffer 2.7 Imaging Buffer 2.8 Preparation of Slides with Microfluidic Channel 2.9 Oligonucleotides 2.10 Microscope and Hardware 2.11 Software 3 Methods 3.1 Preparation of RNA Complex for SiM-KARTS 3.2 Immobilization of Reconstituted preQ1 Riboswitch Containing mRNA Complex onto Microscope Slides 3.3 Data Acquisition 3.4 Data Analysis 3.5 Trace Idealization 3.6 Calculating the Fano Factor 3.7 Calculating the Burst and Dwell Times 4 Conclusions 5 Notes References Chapter 5: Isothermal Titration Calorimetry Analysis of a Cooperative Riboswitch Using an Interdependent-Sites Binding Model 1 Introduction 2 Materials 2.1 Preparation of Receptor and Ligand 2.2 Instrumentation 3 Methods 3.1 Performing the ITC Experiment 3.2 Considerations for Analyzing Parabolic Thermograms 3.3 Analysis of Parabolic Thermograms 3.3.1 Theory Behind the Nonequivalent-Interdependent-Sites Binding Model 3.3.2 Optimization of Model Parameters 3.3.3 Global Fitting Provides Better Coverage of Binding Events 3.3.4 Calculation of Cooperativity 3.4 Application of the Interdependent Sites Model 3.4.1 Preparing ITC Data for Analysis 3.4.2 Running the Python Program 3.5 Interpretation of Output Data 3.6 Code Availability 4 Notes References Chapter 6: Application of Molecular Dynamics to Expand Docking Program´s Exploratory Capabilities and to Evaluate Its Predicti... 1 Introduction 2 Molecular Dynamics Simulations of RNA Receptors 2.1 Use LEaP to Prepare the System for Simulations 2.2 Perform System Equilibration 2.3 Molecular Dynamics Production Run 3 Molecular Dynamics Trajectory Sampling 4 rDock Ligand Docking to RNA Receptors 4.1 Prepare the Ligand SD File 4.2 Prepare RNA Receptor Files in the Tripos MOL2 Format 4.3 Prepare the System Definition File with the Receptor and the Docking Cavity Parameters 4.4 Generate the Docking Site (Cavity) File Using rDock´s rbcavity Module 4.5 Generate Docking Pose Predictions for the Ligand(s) Using rDock´s rbdock Module 4.6 rDock Results Analysis 4.7 Molecular Dynamics Simulations of RNA-Ligand Complexes 5 Notes References Chapter 7: Exploring the Binding Process of Cognate Ligand to Add Adenine Riboswitch Aptamer by Using Explicit Solvent Molecul... 1 Introduction 2 Materials 2.1 Hardware Preparation 2.2 Software Configuration 3 Methods 3.1 Construction of Initial System 3.2 Running Minimization and Molecular Dynamics Simulation 3.2.1 Minimization of the Initial System 3.2.2 Equilibration of the Molecular System 3.2.3 Production MD Simulation of the System 3.3 Trajectory Analysis and Binding Free Energy 3.3.1 Trajectory Analysis 3.3.2 Calculation of Binding Free Energy 4 Notes References Chapter 8: Prediction of Dynamic RBP-RNA Interactions Using PrismNet 1 Introduction 2 PrismNet Install 3 Data Preparation and Generation 4 PrismNet Model Training and Prediction 4.1 Training 4.2 Evaluation 4.3 Inference 5 Saliency Map Generation 5.1 Saliency Map Calculation 5.2 High Attention Regions Calculation 5.3 Plot Image of Saliency Map 6 Motif Construction References Chapter 9: High-Resolution Atomic Force Microscopy Imaging of RNA Molecules in Solution 1 Introduction 2 Materials 3 Methods 3.1 Sample Preparation 3.1.1 Mica Substrate Preparation 3.1.2 Chemical Modification of Mica with APS 3.1.3 RNA Sample Preparation for High-Resolution AFM 3.2 Atomic Force Microscopy 3.2.1 AFM Setup 3.2.2 Image Acquisition 3.2.3 Image Processing 4 Notes References Chapter 10: 3D Structural Analysis of Long Noncoding RNA by Small Angle X-ray Scattering and Computational Modeling 1 Introduction 2 Materials 2.1 DNA Molecular Cloning 2.2 RNA Production by In Vitro Transcription 2.3 RNA Purification by Preparative Chromatography 2.4 RNA Purification by Preparative Native PAGE 2.5 RNA Quantification with UV-Vis Spectroscopy 2.6 Sample and Buffer Preparation for SAXS 2.7 SAXS Data Collection 2.8 SAXS Data Analysis 2.9 SAXS-Aided RNA Structure Prediction and Computational Modeling 2.10 RNA Ensemble Analysis Using the Ensemble Optimization Method 3 Methods 3.1 General Considerations 3.2 DNA Molecular Cloning 3.3 RNA Production by In Vitro Transcription 3.4 RNA Purification by Preparative Chromatography 3.5 RNA Purification by Preparative Native PAGE 3.6 RNA Quantification with UV-Vis Spectroscopy 3.7 Sample and Buffer Preparation for SAXS 3.7.1 General Considerations 3.7.2 RNA Sample Preparation 3.7.3 Buffer Recommendation 3.7.4 Sample and Buffer Matching 3.7.5 Sample Shipments 3.8 SAXS Data Collection 3.9 SAXS Data Analysis 3.10 RNA 3D Structure Prediction and SAXS-Aided Computational Modeling 3.11 Ensemble Analysis Using the Ensemble Optimization Method 4 Notes References Chapter 11: Combining Biophysical Methods for Structure-Function Analyses of RNA in Solution 1 Introduction 2 Materials 2.1 Instrumentation 2.2 In Vitro Transcription (IVT) 2.3 RNA Purification Using Native Polyacrylamide Gel Electrophoresis 2.4 Isothermal Titration Calorimetry (ITC) and Small-Angle X-Ray Scattering (SAXS) 2.5 Atomic Force Microscopy 3 Methods 3.1 In Vitro Transcription (IVT) and RNA Purification 3.2 ITC Experiments 3.2.1 RNA Sample and Ligand Solution Preparation 3.2.2 ITC Data Processing and Singular Value Decomposition (SVD) Analysis 3.3 Structure Calculation Using AFM Particle Topographic Restraints 3.3.1 Preparation of AFM Topographic Restraints and Initial Structure Coordinates 3.3.2 Recapitulate Topological Structure Using Dynamic Fitting Algorithm 3.4 SAXS Data Acquisition and Ensemble Fitting Using AFM-Derived Structures 3.4.1 SAXS Instrument Setup 3.4.2 Sample Preparation for SAXS Experiments 3.4.3 SAXS Data Acquisition and Processing 3.4.4 SAXS Ensemble Fitting Guided by AFM-Derived Structures 4 Notes References Chapter 12: Near-Atomic Resolution Cryo-EM Image Reconstruction of RNA 1 Introduction 2 Materials 2.1 Reagents 2.2 Equipment 2.3 Software 3 Experimental and Computational Methods 3.1 RNA Sample Preparation 3.2 RNA Folding 3.3 Cryo-EM Freezing and Data Collection 3.4 Cryo-EM Image Analysis and Reconstruction 3.4.1 All Micrographs Were Motion-Corrected Using MotionCor2 3.4.2 Contrast Transfer Function (CTF) Was Determined Using CTFFIND4 3.4.3 All Particles Were Autopicked Using the NeuralNet Option in EMAN2 3.4.4 2D Classification in Relion 3.4.5 Map Determination in cryoSPARC 3.5 Map Sharpening and Masking 4 Map Validation 5 Map and Model Submission to wwPDB 6 Notes References Chapter 13: Auto-DRRAFTER: Automated RNA Modeling Based on Cryo-EM Density 1 Introduction 2 Materials 2.1 Recommended Computing Requirement 2.2 Installing Software 2.2.1 Rosetta (auto-DRRAFTER) 2.2.2 Python 2.2.3 UCSF Chimera 2.2.4 EMAN2 2.3 Prepare Input Files 3 Methods 3.1 Auto-DRRAFTER Overview 3.2 Step-by-Step Method Details 3.2.1 Prepare Input Files Including RNA Secondary Structure (Fig. 2a) and Cryo-EM Density Map (Fig. 2b) 3.2.2 Low-Pass Filter the Cryo-EM Density Map and Determine an Appropriate Threshold Level (Fig. 2d) to Find End Nodes (Fig. 2... 3.2.3 Select End Nodes for Probe Helix Placement (Fig. 2d, e) 3.2.4 Build Initial Models with Different End Nodes (Fig. 2f) 3.2.5 Iterative Refinement of RNA Models (Fig. 2f, g) 3.2.6 Final Two Rounds of Modeling (Fig. 2g) 3.2.7 Completing the Modeling Procedure 4 Notes References Chapter 14: X-Ray Crystallography to Study Conformational Changes in a TPP Riboswitch 1 Introduction 2 Materials 2.1 RNA Preparation 2.1.1 DNA Template Preparation 2.1.2 In Vitro Transcription 2.1.3 Polyacrylamide Gel Electrophoresis (PAGE) 2.1.4 RNA Chromatography 2.2 Crystallization 3 Methods 3.1 RNA Preparation 3.1.1 DNA Template Preparation 3.1.2 In Vitro Transcription 3.1.3 Large-Scale PAGE for RNA 3.1.4 RNA Chromatography 3.2 Crystallization 4 Notes References Chapter 15: Preparation of RNA Microcrystals for Serial Femtosecond Crystallography Experiments 1 Introduction 2 Materials 3 Methods 3.1 Initial Growth and Characterization of RNA Microcrystals 3.2 Batch Crystallization Trials 3.3 Optimizing Conditions for Large-Scale Batch Crystallization 3.4 Large-Scale Crystallization of RNA Using the Batch Method 4 Notes References Chapter 16: Mix-and-Inject Serial Femtosecond Crystallography to Capture RNA Riboswitch Intermediates 1 Introduction 2 Materials 3 Methods 3.1 Hardware Preparation for Mix-and-Inject Experiment 3.2 Data Processing and Analysis 4 Notes References Index