UID:
edoccha_9958130548802883
Format:
1 online resource (675 p.)
Edition:
First edition.
ISBN:
0-12-801936-0
,
0-12-801934-4
Series Statement:
Methods in Enzymology ; Volume 558
Content:
This new volume of 〈i〉Methods in Enzymology〈/i〉 continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers research methods in RNA folding and dynamics, RNA-protein interactions and large RNPs.〈br〉〈br〉〈ul〉〈li〉Continues the legacy of this premier serial with quality chapters on structures of large RNA molecules and their complexes〈/li〉〈/ul〉
Note:
Description based upon print version of record.
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Front Cover; Structures of Large RNA Molecules and Their Complexes; Copyright; Contents; Contributors; Preface; Section I: RNA Structure and Dynamics; Chapter 1: Native Purification and Analysis of Long RNAs; 1. Introduction; 2. Native Purification of Long Noncoding RNAs; 2.1. Construct design; 2.2. DNA plasmid linearization; 2.3. In vitro transcription of RNA; 2.4. DNase digestion; 2.5. Proteinase K treatment; 2.6. EDTA chelation of divalent ions (optional); 2.7. Buffer exchange and purification; 2.8. Size-exclusion chromatography
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3. Study of the RNA Tertiary Folding by Sedimentation Velocity Analytical Ultracentrifugation3.1. Preparation of samples for a study of RNA folding; 3.2. Assembly of the optical cells, sample loading, and instrument setup; 3.3. Setting up a sedimentation velocity experiment; 3.4. Data analysis; 4. Analysis of the RNA Tertiary Folding by Analytical Size-Exclusion Chromatography; 5. Determination of the Secondary Structure of LncRNAs by Chemical Probing; 5.1. Designing and coupling of primers; 5.2. Generation of sequencing ladders; 5.3. SHAPE reaction; 5.4. DMS reaction
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5.5. Primer extension reaction5.6. Reactions for mobility shift correction; 5.7. Spectral calibration of the instrument; 5.8. Preparation of samples for capillary electrophoresis; 5.9. Data analysis; 5.9.1. Determination of chemical probing reactivity profiles; 5.9.2. Normalization of SHAPE and DMS reactivity profiles; 5.9.3. RNA secondary structure prediction and analysis; Acknowledgments; References; Chapter 2: Characterizing RNA Excited States Using NMR Relaxation Dispersion; 1. Introduction; 2. NMR Relaxation Dispersion; 2.1. Chemical Exchange; 2.2. RD Experiments
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2.3. R1ρ with Low-to-High SL Fields3. General Protocol for Characterizing RNA ESs Using Low-to-High SL Field 13C and 15N Off-Resonance NMR R1ρ and Uniformly...; 3.1. Construct Design; 3.2. Sample Preparation and Purification; 3.3. Protocol for Measuring R1ρ RD in Uniformly Labeled Nucleic Acids; 3.3.1. Calibrating SL Power; 3.3.2. Measurement of 15N R1ρ Data; 3.3.3. Measurement of 13C R1ρ Data; 3.3.4. Trouble Shooting R1ρ; 3.4. Data Analysis; 3.4.1. Fitting Monoexponentials to Obtain R1ρ Values; 3.4.2. Fitting Off-Resonance R1ρ Data Using Algebraic Equations
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3.4.3. Fitting Off-Resonance R1ρ Data Using Bloch-McConnell Equations3.4.4. Determining the Chemical Shifts of the ES; 3.4.5. Plotting RD Profiles; 3.4.6. Estimating Uncertainties in Exchange Parameters; 3.4.7. Kinetic-Thermodynamic Analysis; 3.5. Inferring Structures of RNA ESs Using NMR Chemical Shifts and Secondary Structure Prediction; 3.5.1. 13C and 15N Chemical Shift-Structure Relationships in RNA; 3.5.2. Secondary Structure Prediction; 3.6. Testing Model RNA ESs; 3.6.1. Stabilizing GS and ES Using Mutations; 3.6.2. Stabilizing GS and ES Using Single-Atom Substitutions
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3.6.3. Stabilizing GS and ES by Changing pH
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English
Language:
English
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