NMR Analysis of Base‐Pair Opening Kinetics in DNA

Marta W. Szulik1, Markus Voehler1, Michael P. Stone2

1 These authors contributed equally to this work, 2 Department of Chemistry and Center for Structural Biology, Vanderbilt University, Nashville, Tennesee
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 7.20
DOI:  10.1002/0471142700.nc0720s59
Online Posting Date:  December, 2014
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Abstract

Base pairing in nucleic acids plays a crucial role in their structure and function. Differences in the base‐pair opening and closing kinetics of individual double‐stranded DNA sequences or between chemically modified base pairs provide insight into the recognition of these base pairs by DNA processing enzymes. This unit describes how to quantify the kinetics for localized base pairs by observing changes in the imino proton signals by nuclear magnetic resonance spectroscopy. The determination of all relevant parameters using state‐of‐the art techniques and NMR instrumentation, including cryoprobes, is discussed. © 2014 by John Wiley & Sons, Inc.

Keywords: base‐pair opening; exchange kinetics; imino‐exchange; longitudinal water relaxation NMR; nucleic acids; T1; high Q‐probes; radiation damping

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Sample Preparation
  • Basic Protocol 2: Measurment of the T1 Relaxation Time of Water Using the Saturation‐Recovery Method
  • Alternate Protocol 1: T1 Relaxation Time of Water Using the Standard Inversion‐Recovery Method
  • Basic Protocol 3: Measurement of the Relaxation R1n for the Imino Protons
  • Basic Protocol 4: Measurement of the Exchange Rate for the Imino Protons
  • Basic Protocol 5: Measurement of the Water Inversion Efficiency Factor (E)
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Sample Preparation

  Materials
  • 10 mM sodium phosphate buffer at desired pH (see recipe for 100 mM sodium phosphate buffer in appendix 2A), prepared using 95% deionized H 2O and 5% D 2O (99.99%)
  • Sodium chloride (NaCl)
  • Disodium EDTA
  • Triethanolamine (TEOA)
  • Ammonium hydroxide (NH 4OH)
  • Hydrochloric acid (HCl)
  • Sodium hydroxide (NaOH)
  • Purified and desalted DNA samples, lyophilized
  • Bath sonicator
  • 0.5‐mL microcentrifuge tubes
  • 3‐mm NMR tubes

Basic Protocol 2: Measurment of the T1 Relaxation Time of Water Using the Saturation‐Recovery Method

  Materials
  • 3‐mm NMR tube containing double‐stranded DNA in NMR buffer with 5% D 2O (see protocol 1)
  • High‐field NMR spectrometer (AV‐III, Bruker Biospin) equipped with quadruple nuclei (1H, 13C, 15N, and 31P) cryogenically cooled probe with z‐gradient
  • TopSpin 3.2 software (Bruker Biospin)

Alternate Protocol 1: T1 Relaxation Time of Water Using the Standard Inversion‐Recovery Method

  Materials
  • 3‐mm NMR tube containing double‐stranded DNA in NMR buffer with 5% D 2O (see protocol 1)
  • Additional reagents and equipment for setting up the spectrometer ( protocol 2)

Basic Protocol 3: Measurement of the Relaxation R1n for the Imino Protons

  Materials
  • 3‐mm NMR tube containing double‐stranded DNA in NMR buffer with 5% D 2O (see protocol 1)
  • GraphPad Prism software (v. 6.0b for Mac OSX; http://www.graphpad.com)
  • Additional reagents and equipment for setting up the spectrometer ( protocol 2) and determining the transmitter offset and π/2 hard pulse ( protocol 4, step 1)

Basic Protocol 4: Measurement of the Exchange Rate for the Imino Protons

  Materials
  • 3‐mm NMR tube containing double‐stranded DNA in NMR buffer with 5% D 2O (see protocol 1)
  • Additional reagents and equipment for setting up the spectrometer (Basic Protocol protocol 5)
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Figures

Videos

Literature Cited

Literature Cited
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