Determination of equilibrium binding affinity of distamycin and netropsin to the synthetic deoxyoligonucleotide sequence d(GGTATACC)2 by quantitative DNase I footprinting
- 1 August 1988
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 27 (16) , 6026-6032
- https://doi.org/10.1021/bi00416a030
Abstract
A new method for determining the equilibrium binding constant of antitumor drugs to specific DNA sequences by quantitative DNase I footprinting is presented. The use of a short synthetic DNA oligomer to define a homogeneous population of DNA binding sites enables the calculation of the free drug concentration and the fraction of DNA sites complexed with drug in solution and is described for the first time. Since a 1:1 stoichiometry is observed for each drug-oligomer DNA complex, it becomes possible to calculate equilibrium binding constants in solution. By use of this technique, the binding affinities of the nonintercalating drugs netropsin and distamycin to the synthetic oligonucleotide d(GGTATACC)2 are determined to be Ka(25.degree. C) = 1.0 .times. 105 and 2.0 .times. 105 M-1, respectively. Quantitation of the temperature dependence associated with complex formation results in a determination of standard enthalpies of -3.75 and -8.48 kcal mol-1 for the binding of netropsin and distamycin, respectively. Calculation of other thermodynamic parameters are found to be in agreement with previous studies and indicate that the DNA binding process for these compounds is predominantly enthalpy driven. This method of quantitative DNase I footprinting is demonstrated to be a useful technique for the measurement of drug affinities to specific binding sites on DNA oligomers which are designed and synthesized expressly for this purpose. Applications of the technique to the determination of drug binding affinities at specific sites within native DNA sequences are discussed.This publication has 12 references indexed in Scilit:
- The molecular origin of DNA-drug specificity in netropsin and distamycin.Proceedings of the National Academy of Sciences, 1985
- Single-cell partition analysis—A direct fluorescence technique for examining ligand-macromolecule interactionsAnalytical Biochemistry, 1983
- Sequence specificity of actinomycin D and Netropsin binding to pBR322 DNA analyzed by protection from DNase I.Proceedings of the National Academy of Sciences, 1983
- Kinetics for exchange of the imino protons of the d(C-G-C-G-A-A-T-T-C-G-C-G) double helix in complexes with the antibiotics netropsin and/or actinomycinBiochemistry, 1983
- Mutual interaction between adjacent dG . dC actinomycin binding sites and dA . dT netropsin binding sites on the self-complementary d(C-G-C-G-A-A-T-T-C-G-C-G) duplex in solution.Proceedings of the National Academy of Sciences, 1981
- Structure of a B-DNA dodecamerJournal of Molecular Biology, 1981
- Magnetic circular dichroism study of the binding of netropsin and distamycin-A with DNABiochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis, 1980
- Netropsin · dG‐dG‐dA‐dA‐dT‐dT‐dC‐dC ComplexEuropean Journal of Biochemistry, 1979
- Netropsin, a DNA-binding oligopeptide structural and binding studiesBiochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis, 1979
- Theoretical calculations of the helix–coil transition of DNA in the presence of large, cooperatively binding ligandsBiopolymers, 1976