SPECIFICITY OF INTERCALATION REACTIONS

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The long-range objective of the proposed research is to understand the molecular mechanisms that govern the sequence-specific DNA binding of clinically useful intercalating antibiotics. A quantitative DNase I footprinting titration method will be used to identify the preferred DNA binding sites of the anticancer anthracycline antibiotics (daunomycin, adriamycin, nogalamycin) and the newly synthesized anthrapyrazole antibiotics. The method will further be used to estimate binding constants for the interaction of these compounds with their preferred sites. Once the identity of these preferred sites is known, more detailed binding studies using synthetic deoxyoligonucleotides of designed sequence will be conducted, using the methods of absorbance and fluorescence spectroscopy, titration calorimetry, and stopped-flow kinetics. These studies will provide a detailed thermodynamic and kinetic profile describing the specific binding of intercalators to their preferred DNA sites, a fundamental characterization that has heretofore been lacking. This information will complement existing structural data for the anthracycline antibiotics obtained by x-ray diffraction, and should help identify the molecular determinants of site specific binding by these compounds. Further, these results should provide a critical test of the predictions derived from molecular mechanics calculations concerning anthracycline antibiotic sequence specificity. Finally, the possible linkage between sequence specific antibiotic binding and topoisomerase II inhibition will be explored by DNase I footprinting methods. All of the proposed studies are logical extensions of what have been successful, productive and cost-effective research efforts in our laboratory. The results of these studies should provide fundamental physical chemical data of possible use in the rational design of new intercalating antibiotics targeted toward specific DNA binding sites.