Detection of Hydrogen Bonds in Dynamic Regions of RNA by NMR Spectroscopy

Andre Dallmann1, Michael Sattler2

1 Present address: Division of Molecular Structure, National Institute of Medical Research, London, 2 Center for Integrated Protein Science Munich and Chair of Biomolecular NMR‐Spectroscopy, Department Chemie, Technische Universität München, Garching
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 7.22
DOI:  10.1002/0471142700.nc0722s59
Online Posting Date:  December, 2014
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Abstract

NMR spectroscopy is a powerful tool to study the structure and dynamics of nucleic acids. In this unit, we give an overview of important experiments to determine and characterize hydrogen bonds in nucleic acids and provide detailed instructions for setting up recently developed sensitivity‐improved NMR pulse sequences, i.e., BEST selective long‐range HNN‐COSY, selective BEST‐TROSY‐HNNCOSY, and Py H(CC)NN‐COSY. The strengths and limitations of these experiments will also be discussed. Detailed step‐by‐step protocols are provided for each of the three pulse sequences, with special emphasis on adjusting and setting of delays and shaped pulses. The NMR pulse sequences with example datasets and optimized, nonstandard adiabatic pulse shapes used for selective 15N magnetization transfer are provided. These experiments enable NMR analysis of a broad variety of RNAs ranging from low to high molecular weight and complexity. © 2014 by John Wiley & Sons, Inc.

Keywords: NMR spectroscopy; hydrogen bonds; dynamics; RNA; sensitivity enhancement

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Best Selective Long‐Range HNN‐COSY (BESTsellr)
  • Basic Protocol 2: Best TROSY‐HNN‐COSY
  • Basic Protocol 3: Pyrimidine (Py) H(CC)NN‐COSY
  • Commentary
  • Figures
  • Tables
     
 
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Materials

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Figures

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Literature Cited

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Key Reference
  Dallmann et al., 2013. See above.
  This is the original publication of the BESTsellr HNN‐COSY and Py H(CC)NN‐COSY experiments, including more theoretical background on the pulse sequences. The protocols presented here focus on practical aspects of the implementation of these experiments.
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