Characterization of Quadruplex DNA Structure by Circular Dichroism

Rafael del Villar‐Guerra1, Robert D. Gray1, Jonathan B. Chaires1

1 James Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 17.8
DOI:  10.1002/cpnc.23
Online Posting Date:  March, 2017
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Abstract

Circular dichroism (CD) is a phenomenon that arises from the differential absorption of left‐ and right‐handed circularly polarized light, and may be seen with optically active molecules. CD spectroscopy provides useful spectral signatures for biological macromolecules in solution, and provides low‐resolution structural information about macromolecular conformation. CD spectroscopy is particularly useful for monitoring conformational changes in macromolecules upon environmental perturbations. G‐quadruplex structures show unique CD spectral signatures, and CD is an important tool for characterizing their formation and global structure. This protocol offers step‐by‐step methods for determining reliable and reproducible CD spectra of quadruplex structures and normalizing the spectra for presentation. CD spectra properly normalized with respect to quadruplex concentration and path length are required to facilitate accurate comparison of results among laboratories. The standard operating procedures proposed are recommended to make such comparison accurate and informative. © 2017 by John Wiley & Sons, Inc.

Keywords: DNA; G‐quadruplex; circular dichroism; concentration determination

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Measuring CD and Absorbance Spectra of G‐Quadruplexes
  • Support Protocol 1: Preparation of Folded G‐Quadruplex Solutions
  • Support Protocol 2: Determination of the Molar Extinction Coefficient of a Folded G‐Quadruplex Using Alkaline Denaturation
  • Support Protocol 3: Determination of the Molar Extinction Coefficient of a Folded G‐Quadruplex Using Thermal Denaturation
  • Support Protocol 4: Determination of Folded G‐Quadruplex Concentration by Absorbance Spectroscopy
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measuring CD and Absorbance Spectra of G‐Quadruplexes

  Materials
  • Folded G‐quadruplex solution with total A 260 of 0.8‐1.2 in a 1‐cm‐pathlength cuvette (see protocol 2)
  • Folding buffer (see Critical Parameters and Troubleshooting)
  • Jasco J‐810 CD spectropolarimeter with programmable Peltier‐thermostatted cell holder and stirrer
  • UV/visible spectrophotometer with temperature‐controlled cell holder
  • Stoppered quartz cuvette with 0.01‐ or 1‐cm‐pathlength (depending on DNA strand concentration)
  • Origin software (OriginLab)

Support Protocol 1: Preparation of Folded G‐Quadruplex Solutions

  Materials
  • Lyophilized DNA oligonucleotide with standard desalting
  • Folding buffer (see Critical Parameters and Troubleshooting)
  • Screw‐cap plastic microcentrifuge tubes
  • 1 liter boiling water bath

Support Protocol 2: Determination of the Molar Extinction Coefficient of a Folded G‐Quadruplex Using Alkaline Denaturation

  Materials
  • Folded G‐quadruplex solution with total A260 of 0.8‐1.2 in a 1‐cm‐pathlength cuvette (see protocol 2)
  • 5 M NaOH solution
  • Folding buffer (see Critical Parameters and Troubleshooting)
  • UV/visible spectrophotometer with temperature‐controlled cell holder
  • 1‐cm‐pathlength stoppered quartz cuvette
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Figures

Videos

Literature Cited

Literature Cited
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