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

项目摘要 提出的研究目标是研究两者之间的结构-功能关系 三链DNA和一类新发现的基于黄酮类的三链特异结合配体及其用途 获得设计和开发更有效的三链结合配体作为抗基因的知识 疾病治疗抗基因策略中的增强剂。这项建议可分为三类 部分，结构修饰(合成)，用生物物理方法测定配体-DNA相互作用 方法，并进行了抑制酶活性的研究。合成项目包括改变 连接基长度，去掉官能团，并延伸芳香表面。每种结构 修改意在解决对结构-功能关系的具体询问。这个 合成过程涉及基本的有机技术。合成的分子随后将被用于 用不同的生物物理方法研究它们与几种DNA结构的相互作用。稳定化 三链和双链DNA与配体在不同条件下(pH，离子强度)将 用UC监测的热变性测定。DNA和DNA的构象变化 结合化学计量比将使用圆二向色性进行测量。与DNA的选择性结合 将在相对较高的吞吐量下使用竞争透析来确定特定构象 时尚。配体-三链DNA络合物的转变热将直接用 差示扫描量热法(DSC)。热力学参数，包括热焓， 用等温滴定量热法测定熵、自由能和结合位大小 (ITC)。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。 得出的结论。完成生物物理研究后，具有最佳结合强度的配体 生化反应将选择特异性。一种含有三链形成的质粒DNA 网站将被使用。在该质粒dna中，存在四个限制性内切酶dri裂解位点。 其中一个残基正好位于三链螺旋的交界处。形成了一种 三链DNA应该保护Drai裂解位点，导致DRAI裂解位点的消失和出现 具有特定长度的产物，通过琼脂糖凝胶电泳法可以很容易地检测到。这个 拟议工作的结果将是科学界第一次努力理解这些 新发现的三链特异结合配体。研究的性质是适合培训的 在一家以本科生为重点的机构的本科生和研究生研究员。

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.