Determination of Binding Thermodynamics

Peter C. Dedon1

1 Massachusetts Institute of Technology, Cambridge, Massachusetts
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 8.2
DOI:  10.1002/0471142700.nc0802s00
Online Posting Date:  May, 2001
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Abstract

This unit serves as a starting point for exploring the thermodynamic properties of interactions between small molecules and DNA. It covers the determination of simple, apparent association/dissociation constants. The concentration of DNA‐bound ligand and free ligand are determined and a binding constant is extracted from these data. Data gathering and curve fitting are discussed.

     
 
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Table of Contents

  • Basic Protocol 1: Spectroscopic Titration of Fixed DNA Concentrations with Varying Ligand Concentrations
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Spectroscopic Titration of Fixed DNA Concentrations with Varying Ligand Concentrations

  Materials
  • Assay buffer: 10 mM Tris⋅Cl, HEPES, or other buffer, pH 7, containing 1 mM EDTA
  • Calf thymus DNA, sonicated (see recipe)
  • Ligand of interest dissolved in assay buffer
  • UV/vis spectrophotometer with temperature‐controlled cuvette holder
  • 1‐mL cuvette
  • Software capable of linear regression analysis (e.g., Microsoft Excel)
  • Additional reagents and equipment for determination of extinction coefficients of free and DNA‐bound ligands (unit 8.1, protocol 5)
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Figures

Videos

Literature Cited

Literature Cited
   Chaires, J.B. 1992. Application of equilibrium binding methods to elucidate the sequence specificity of antibiotic binding to DNA. In Advances in DNA Sequence Specific Agents, Vol. 1 (L. Hurley, ed.) pp. 3‐23. JAI Press, New York.
   Chaires, J.B., Dattagupta, N., and Crothers, D.M. 1982. Studies on interaction of anthracycline antibiotics and deoxyribonucleic acid: Equilibrium binding studies on interaction of daunomycin with deoxyribonucleic acid. Biochemistry 21:3933‐3940.
   Correia, J.J. and Chaires, J.B. 1994. Analysis of drug‐DNA binding isotherms: A Monte Carlo approach. Methods Enzymol. 240:593‐614.
   Dattagupta, N., Hogan, M., and Crothers, D.M. 1980. Interaction of netropsin and distamycin with deoxyribonucleic acid: Electric dichroism study. Biochemistry 19:5998‐6005.
   Hinton, D.M. and Bode, V.C. 1975. Ethidium bromide affinity of circular λ‐deoxyribonucleic acid determined fluorimetrically: The effect of NaCl concentration on supercoiling. J. Biol. Chem. 250:1061‐1070.
   Johnson, M.L. and Faunt, L.M. 1992. Parameter estimation by least‐squares methods. Methods Enzymol. 210:1‐37.
   Krishnamurthy, G., Brenowitz, M.D., and Ellestad, G.A. 1995. Salt‐dependence of calicheamicin‐DNA site‐specific interactions. Biochemistry 34:1001‐1010.
   McGhee, J.D. and von Hippel, P.H. 1974. Theoretical aspects of DNA‐protein interactions: Co‐operative and non‐co‐operative binding of large ligands to a one‐dimensional homogeneous lattice. J. Mol. Biol. 86:469‐489.
   Minton, A.P. 1990. Quantitative characterization of reversible molecular associations via analytical centrifugation. Anal. Biochem. 190:1‐6.
   Record, M.T. Jr., Anderson, C.F., and Lohman, T.M. 1978. Thermodynamic analysis of ion effects on the binding and conformational equilibria of proteins and nucleic acids: The role of ion association or release, screening, and ion effects on water activity. Q. Rev. Biophys. 11:103‐178.
   Waring, M.J. 1965. Complex formation between ethidium bromide and nucleic acids. J. Mol. Biol. 13:269‐282.
   Waring, M.J., Wakelin, L.P.G., and Lee, J.S. 1975. A solvent‐partition method for measuring the binding of drugs to DNA: Application to the quinoxaline antibiotics echinomycin and triostin A. Biochim. Biophys. Acta 407:200‐212.
   Yu, L., Golik, J., Harrison, R., and Dedon, P. 1994. The deoxyfucose‐anthranilate of esperamicin A1 confers intercalative DNA binding and causes a switch in the chemistry of bistranded DNA lesions. J. Am. Chem. Soc. 116:9733‐9738.
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