Calorimetry of Nucleic Acids

Eriks Rozners1, Daniel S. Pilch2, Martin Egli3

1 Binghamton University, State University of New York, Binghamton, New York, 2 Rutgers Robert Wood Johnson Medical School, Piscataway, New Jersey, 3 Vanderbilt University, Nashville, Tennessee
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 7.4
DOI:  10.1002/0471142700.nc0704s63
Online Posting Date:  December, 2015
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Abstract

This unit describes the application of calorimetry to characterize the thermodynamics of nucleic acids, specifically, the two major calorimetric methodologies that are currently employed: differential scanning (DSC) and isothermal titration calorimetry (ITC). DSC is used to study thermally induced order‐disorder transitions in nucleic acids. A DSC instrument measures, as a function of temperature (T), the excess heat capacity of a nucleic acid solution relative to the same amount of buffer solution. From a single curve of versus T, one can derive the following information: the transition enthalpy (ΔH), entropy (ΔS), free energy (ΔG), and heat capacity (ΔCp); the state of the transition (two‐state versus multistate); and the average size of the molecule that melts as a single thermodynamic entity (e.g., the duplex). ITC is used to study the hybridization of nucleic acid molecules at constant temperature. In an ITC experiment, small aliquots of a titrant nucleic acid solution (strand 1) are added to an analyte nucleic acid solution (strand 2), and the released heat is monitored. ITC yields the stoichiometry of the association reaction (n), the enthalpy of association (ΔH), the equilibrium association constant (K), and thus the free energy of association (ΔG). Once ΔH and ΔG are known, ΔS can also be derived. Repetition of the ITC experiment at a number of different temperatures yields the ΔCp for the association reaction from the temperature dependence of ΔH. © 2015 by John Wiley & Sons, Inc.

Keywords: differential scanning calorimetry; isothermal titration calorimetry; enthalpy; entropy; heat capacity

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Differential Scanning Calorimetry of Nucleic Acids
  • Basic Protocol 2: Isothermal Titration Calorimetry of Nucleic Acids
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Differential Scanning Calorimetry of Nucleic Acids

  Materials
  • Appropriate buffer
  • Buffer solution of purified nucleic acid (0.2 to 2.0 mM in nucleotide)
  • Nitrogen (N 2) gas
  • Differential scanning calorimetry (DSC) instrument
  • Vacuum source and side‐arm flask (for degassing)
  • Float‐A‐Lyzer G2 1 mL dialysis bag (e.g., Spectrum Laboratories)

Basic Protocol 2: Isothermal Titration Calorimetry of Nucleic Acids

  Materials
  • Appropriate buffer
  • Buffer containing purified analyte nucleic acid (nucleic acid A; 0.1 to 1.0 mM in nucleotide depending on expected K)
  • Buffer containing purified titrant nucleic acid (nucleic acid T), whose base sequence is complementary to that of nucleic acid A and whose concentration is 10 times that of nucleic acid A
  • Isothermal titration calorimetry (ITC) instrument
  • Vacuum source and side‐arm flask (for degassing)
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Figures

Videos

Literature Cited

Literature Cited
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