Development of flavone- and flavonoid-based DNA triplex specific binding ligands as antigene enhancers

项目摘要 本研究的主要目的是研究蛋白质结构与功能的关系， 三链体DNA和一类新发现的类黄酮基三链体特异性结合配体及其用途 所获得的知识设计和开发更有效的三链体结合配体作为抗原 在疾病治疗的抗基因策略的增强剂。这项建议可分为三种 部分，结构修饰（合成），通过生物物理方法测定配体-DNA相互作用 方法和酶活性抑制的研究。综合项目包括改变 连接基长度、除去官能团和延伸芳族表面。每个结构 修改旨在解决对结构-功能关系的具体询问。的 合成过程涉及基本的有机技术。合成的分子将被用来 通过各种生物物理方法研究它们与几种DNA结构的相互作用。稳定 三链体和双链体DNA与配体在各种条件下（pH，离子强度）的反应将被 使用由UC监测的热变性测定。DNA的构象变化， 使用圆二色性测量结合化学计量。选择性结合DNA， 将使用竞争透析以相对高的通量测定特定的构象 时尚.配体-三链体DNA复合物的转变焓将使用以下直接测量： 差示扫描量热法（DSC）。热力学参数，包括焓， 熵、自由能和结合位点大小将使用等温滴定量热法测定 （国际贸易中心）。值得注意的是，生物物理研究的结果将用于交叉检查，以确保准确性 的结论。在完成生物物理研究后，具有最佳结合强度的配体 并且选择特异性用于生化反应。含有三链体形成的质粒DNA， 网站将被使用。在该质粒DNA中，存在四个限制性内切酶DraI切割位点， 质粒，其中一个残基正好位于三链体螺旋位点的连接处。的形成 三链DNA应该保护DraI切割位点，导致 具有特定长度的产物，其可以容易地通过琼脂糖凝胶电泳检测。的 从拟议的工作结果将是第一次努力，在科学界了解这些 新发现的三链体特异性结合配体。研究的性质适合培训 本科生和研究生研究人员在本科为重点的机构。

Project Summary The proposed research objectives are to study the structure-function relationship between triplex DNA and a class of newly discovered flavonoid-based triplex-specific binding ligands and use the obtained knowledge to design and develop more potent triplex binding ligands as antigene enhancers in the antigene strategy for disease treatment. This proposal can be categorized into three portions, structural modification (synthesis), determination of ligand-DNA interactions by biophysical methods, and study of inhibition of enzymatic activities. The synthesis project includes changing the linker length, removing the functional groups, and extending the aromatic surface. Each structural modification is intended to address a specific inquiry on the structure-function relationship. The synthesis procedures involve basic organic techniques. The synthesized molecules will then be used to study their interactions with several DNA structures by various biophysical methods. The stabilization of triplex and duplex DNA with ligands under various conditions (pH, ionic strength) will be determined using thermal denaturation monitored by UC. The conformation changes of the DNA and binding stoichiometry will be measured using circular dichroism. Selective binding to DNAs with specific conformations will be determined using competition dialysis in a relatively high throughput fashion. The transition enthalpy of the ligand-triplex DNA complex will be directly measured using differential scanning calorimetry (DSC). The thermodynamics parameters, including enthalpy, entropy, free energy, and binding site size, will be determined using isothermal titration calorimetry (ITC). Notably, results from biophysical studies will be used for cross-inspection to ensure the accuracy of the conclusions. After completion of the biophysical studies, ligands with the best binding strength and specificity will be chosen for biochemical reactions. A plasmid DNA containing a triplex-forming site will be used. In this plasmid DNA, four restriction endonuclease DraI cleavage sites are present in the plasmid, one of which residues exactly at the junction of the triplex helix site. The formation of triplex DNA should protect the DraI cleavage site, leading to the disappearance and appearance of products with specific lengths, which can be readily detected by the agarose gel electrophoresis. The results from the proposed work will be the first effort in the scientific community to understand these newly discovered triplex-specific binding ligands. The nature of the research is suitable for training undergraduate and graduate researchers at an undergraduate-focused institution.