Characterization of Conformational Dynamics of Bistable RNA by Equilibrium and Non‐Equilibrium NMR

Boris Fürtig1, Anke Reining1, Florian Sochor1, Eva Marie Oberhauser2, Alexander Heckel2, Harald Schwalbe1

1 Institute of Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance, Johann Wolfgang Goethe University, Frankfurt, Germany, 2 Institute of Organic Chemistry and Chemical Biology, Cluster of Excellence Macromolecular Complexes, Johann Wolfgang Goethe University, Frankfurt, Germany
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 11.13
DOI:  10.1002/0471142700.nc1113s55
Online Posting Date:  December, 2013
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Abstract

ABSTRACT

Unlike proteins, a given RNA sequence can adopt more than a single conformation. The two (or more) conformations are long‐lived and have similar stabilities, but interconvert only slowly. Such bi‐ or multistability is often linked to the biological functions of the RNA. This unit describes how nuclear magnetic resonance (NMR) spectroscopy can be used to characterize the conformational dynamics of bistable RNAs. Curr. Protoc. Nucleic Acid Chem. 55:11.13.1‐11.13.16. © 2013 by John Wiley & Sons, Inc.

Keywords: bistable RNA; RNA folding; real‐time NMR; exchange spectroscopy; photo‐caged RNA

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Determining the Thermodynamics of Bistable RNA by NMR
  • Basic Protocol 2: Determining the Kinetics of Bistable RNA by Exchange Spectroscopy
  • Alternate Protocol 1: Determining the Kinetics of Bistable RNA by RT‐NMR
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Determining the Thermodynamics of Bistable RNA by NMR

  Materials
  • RNA of interest (≥50 nmol)
  • Low‐salt NMR buffer: 20 to 50 mM potassium phosphate, pH 6.2‐6.5, with 20 to 100 mM KCl in 10% D 2O
  • 5‐mm standard NMR sample tube or NMR microtube with matched magnetic susceptibility
  • NMR high‐field spectrometer equipped with z‐axis gradient 1H{13C,15N} or 1H{13C,31P} triple‐resonance cryogenic probes

Basic Protocol 2: Determining the Kinetics of Bistable RNA by Exchange Spectroscopy

  Materials
  • RNA of interest containing 15N and/or 13C‐labeled sites (≥200 nmol)
  • Low‐salt NMR buffer: 20 to 50 mM potassium phosphate, pH 6.2‐6.5, with 20 to 100 mM KCl in 10% D 2O
  • 5‐mm standard NMR sample tube or NMR microtube with matched magnetic susceptibility
  • NMR high‐field spectrometer equipped with z‐axis gradient 1H{13C,15N} or 1H{13C,31P} triple‐resonance cryogenic probes

Alternate Protocol 1: Determining the Kinetics of Bistable RNA by RT‐NMR

  Materials
  • RNA of interest (≥50 nmol)
  • Low‐salt NMR buffer: 20 to 50 mM potassium phosphate, pH 6.2‐6.5, with 20 to 100 mM KCl in 10% D 2O, or 25 mM potassium arsenate in 10% D 2O
  • 5‐mm pencil‐shaped NMR microtube with matched magnetic susceptibility
  • NMR high‐field spectrometer equipped with z‐axis gradient 1H{13C,15N} or 1H{13C,31P} triple‐resonance cryogenic probes
  • CW argon ion laser (e.g., BeamLok 2060, Spectra Physics, with ∼9 W output power at 340 to 365 nm)
  • Optical fiber (e.g., LWL‐cable UV1000/1100N, Ceram Optec)
  • High‐power resistant lens (e.g., U13×, Newport) mounted in a combined lens and fiber holder (e.g., F‐91‐C1‐T, FPR1‐C1A, Newport)
  • Laser optics such as mirrors and beam splitters (e.g., 200‐0 and 200‐10, CVI)
  • Beam shutter and shutter driver (e.g., Uniblitz electronic VS14S2ZM1 and Uniblitz VCM‐D1, Vincent Associates)
  • Optical breadboard (e.g., MB3090, Thorlabs)
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Figures

Videos

Literature Cited

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