Calorimetry of Nucleic Acids

Daniel S. Pilch1

1 University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 7.4
DOI:  10.1002/0471142700.nc0704s00
Online Posting Date:  May, 2001
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Abstract

This unit describes the application of differential scanning and isothermal titration calorimetry (DSC and ITC) to the study of the thermodynamics of nucleic acid structure. DSC is used to study order‐disorder transitions. A single DSC profile provides a wealth of thermodynamic and extrathermodynamic information: transition enthalpy, entropy, free energy, heat capacity, the state of the transition (two‐state vs. multistate), and the size of the cooperative unit. ITC is used to study hybridization of nucleic acids at constant temperature. Results can be used to determine the stoichiometry of the association reaction, the enthalpy of association, the equilibrium association constant, and the free energy and entropy of association. A thorough discussion is presented of the details required to obtain meaningful results, as well as relevant methods for analyzing the data produced.

     
 
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Table of Contents

  • 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)

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)
  • Buffer containing purified titrant nucleic acid (nucleic acid T), whose base sequence is complementary to that of nucleic acid A and whose concentration is ≥20 times that of nucleic acid A
  • Isothermal titration calorimetry (ITC) instrument
  • Vacuum source and side‐arm flask (for degassing)
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Figures

  •   FigureFigure 7.4.1 DSC profile for the thermal denaturation of the d(CCTCTCCGGCTCTTC)⋅d(GAAGAGCCGGAGAGG) duplex. This DSC measurement was conducted on a model 5100 Nano Differential Scanning Calorimeter (Calorimetry Sciences) using a temperature scanning rate of 60°C/hr. The DNA concentration was 50 µM in duplex and the solution conditions were 10 mM sodium cacodylate (pH 7.0), 100 mM NaCl, 10 mM MgCl2, and 0.1 mM EDTA.
  •   FigureFigure 7.4.2 (A) ITC profile for the hybridization of d(CGTGTCCAGC) and d(GCTGGACACG) at 20°C. This ITC measurement was conducted on a MicroCal model MCS Titration Calorimeter (MicroCal). Five‐microliter aliquots of a d(CGTGTCCAGC) solution (233.5 µM in strand) were sequentially injected from a 100‐µL rotating syringe (400 rpm) into 1.31 mL of a d(GCTGGACACG) solution (8.4 µM in strand). The duration of each injection was 4.93 sec and the delay between injections was 200 sec. The solution conditions were 10 mM sodium cacodylate (pH 7.0), 10 mM KCl, 10 mM MgCl2, and 5 mM CaCl2. (B) Integrated areas of each heat burst curve in (A) plotted as a function of the molar ratio of d(CGTGTCCAGC) to d(GCTGGACACG). The solid line reflects the nonlinear least squares fit of the data to Equation , where K = 6.1 × 107 M–1 and Δ H = –52 kcal/mole.

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Literature Cited

Literature Cited
   Breslauer, K.J. 1995. Extracting thermodynamic data from equilibrium melting curves for oligonucleotide order‐disorder transitions. Methods Enzymol. 259:221‐242.
   Breslauer, K.J., Freire, E., and Straume, M. 1992. Calorimetry: A tool for DNA and ligand‐DNA studies. Methods Enzymol. 211:533‐567.
   Edsall, J.T. and Gutfreund, H. 1983. Calorimetry, heat capacity, and phase transitions. In Biothermodynamics: The Study of Biochemical Processes at Equilibrium, pp. 210‐227. John Wiley & Sons, New York.
   Freire, E., Mayorga, O.L., and Straume, M. 1990. Isothermal titration calorimetry. Anal. Chem. 62:950A‐959A.
   Gralla, J. and Crothers, D.M. 1973. Free energy of imperfect nucleic acid helices III. Small internal loops resulting from mismatches. J. Mol. Biol. 78:301‐319.
   Kamiya, M., Torigoe, H., Shindo, H., and Sarai, A. 1996. Temperature dependence and sequence specificity of DNA triplex formation: An analysis using isothermal titration calorimetry. J. Am. Chem. Soc. 118:4532‐4538.
   Krug, R.R., Hunter, W.G., and Grieger, R.A. 1976. Enthalpy‐entropy compensation. 1. Some fundamental statistical problems associated with the analysis of van't Hoff and Arrhenius data. J. Phys. Chem. 80:2335‐2341.
   Marky, L.A. and Breslauer, K.J. 1987. Calculating thermodynamic data for transitions of any molecularity from equilibrium melting curves. Biopolymers 26:1601‐1620.
   Naghibi, H., Tamura, A., and Sturtevant, J.M. 1995. Significant discrepencies between van't Hoff and calorimetric enthalpies. Proc. Natl. Acad. Sci. U.S.A. 92:5597‐5599.
   Plum, G.E. and Breslauer, K.J. 1995. Calorimetry of proteins and nucleic acids. Curr. Opin. Struct. Biol. 5:682‐690.
   Plum, G.E., Pilch, D.S., Singleton, S.F., and Breslauer, K.J. 1995. Nucleic acid hybridization: Triplex stability and energetics. Annu. Rev. Biophys. Biomol. Struct. 24:319‐350.
   Sturtevant, J.M. 1987. Biochemical applications of differential scanning calorimetry. Annu. Rev. Phys. Chem. 38:463‐488.
   Vesnaver, G. and Breslauer, K.J. 1991. The contribution of DNA single‐stranded order to the thermodynamics of duplex formation. Proc. Natl. Acad. Sci. U.S.A. 88:3569‐3573.
   Wilson, W.D., Hopkins, H.P., Mizan, S., Hamilton, D.D., and Zon, G. 1994. Thermodynamics of DNA triplex formation in oligomers with and without cytosine bases: Influence of buffer species, pH, and sequence. J. Am. Chem. Soc. 116:3607‐3608.
   Wiseman, T., Williston, S., Brandts, J.F., and Lin, L.‐N. 1989. Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal. Biochem. 179:131‐137.
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