SPECIFICITY OF INTERCALATION REACTIONS

拟议研究的长期目标是了解 控制序列特异性DNA结合的分子机制 临床上有用的插层抗生素。一种定量DNA酶I 将使用足迹滴定法来确定首选的DNA 抗癌蒽环类抗生素(柔红霉素， 阿霉素、新霉素)和新合成的蒽普拉唑 抗生素。该方法将进一步用于估计结合常数 用于这些化合物与其首选位置的相互作用。一次 这些首选站点的身份是已知的，更详细的绑定 使用设计序列的合成脱氧寡核苷酸的研究将被 使用吸光度和荧光光谱的方法进行， 滴定量热法和停流动力学。这些研究将 提供详细的热力学和动力学配置文件，描述 嵌入物与其首选DNA位点的特异性结合，a 到目前为止一直缺乏的基本特征。这 信息将补充现有的蒽环类药物的结构数据 通过x射线衍射获得的抗生素，应该有助于识别 这些化合物结合位点特异性的分子决定因素。 此外，这些结果应该会为预测提供一个关键的测试 源自与蒽环类药物有关的分子力学计算 抗生素序列特异性。最后，两者之间可能存在的联系 序列特异性抗生素结合和拓扑异构酶II抑制将 通过DNase I足迹方法进行探索。所有拟议的研究 是对成功的、多产的和 我们实验室的高性价比研究成果。这些研究的结果 研究应提供可能使用的基本物理化学数据 在合理设计新的靶向插层抗生素 特定的DNA结合位点。

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.