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

开发基于黄酮和类黄酮的 DNA 三链体特异性结合配体作为反基因增强剂

• 批准号: 10579779
• 负责人: Liang Xue
• 金额: $ 29.73万
• 依托单位: UNIVERSITY OF THE PACIFIC-STOCKTON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-21 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10579779
• 关键词: Address; Agar Gel Electrophoresis; Appearance; Binding; Binding Sites; Biochemical Reaction; Calorimetry; Chlorides; Circular Dichroism; Communities; Complex; DNA; DNA Binding; DNA Restriction Enzymes; DNA Structure; Development; Dialysis procedure; Differential Scanning Calorimetry; Disease; Enhancers; Ensure; Entropy; Flavones; Flavonoids; Free Energy; Gel; Goals; Institution; Ionic Strengths; Kinetics; Knowledge; Lead; Length; Ligand Binding; Ligands; Measurement; Measures; Modification; Molecular Conformation; Molecular Weight; Monitor; Nature; Oligonucleotides; Pathogenicity; Pharmaceutical Preparations; Plasmids; Procedures; Property; Quercetin; Reporting; Research; Research Personnel; Scheme; Site; Specificity; Structure; Structure-Activity Relationship; Surface; Techniques; Thermodynamics; Titrations; Training; Work; antigene; base; biophysical analysis; biophysical techniques; design; endonuclease; enthalpy; functional group; intercalation; plasmid DNA; scaffold; small molecule; stoichiometry; triplex DNA; undergraduate student

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.

项目摘要 拟议的研究目标是研究 Triplex DNA和一类新发现的基于类黄酮的三重三体特异性结合配体并使用 获得的知识以设计和发展更有效的三重结合配体作为抗基因 抗疾病治疗抗原策略的增强剂。该建议可以分为三个 部分，结构修饰（合成），测定生物物理的配体-DNA相互作用 方法和抑制酶活性的研究。合成项目包括更改 接头长度，删除官能团，并扩展芳香族表面。每个结构 修改旨在解决有关结构功能关系的特定查询。这 合成程序涉及基本的有机技术。然后，合成分子将用于 通过各种生物物理方法研究他们与多种DNA结构的相互作用。稳定 在各种条件下（pH，离子强度）的三链体和双链DNA的配体将是 使用UC监测的热变性确定。 DNA和 结合化学计量将使用圆形二色性测量。选择性结合与DNA的结合 特定构象将使用竞争透析以相对较高的吞吐量确定 时尚。配体三位型DNA复合物的过渡焓将直接使用 差异扫描量热法（DSC）。热力学参数，包括焓， 熵，自由能和结合位点的大小将使用等温滴定量热法确定 （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 结论。完成生物物理研究后，具有最佳结合强度的配体 并将选择特异性用于生化反应。含有三元形成的质粒DNA 站点将被使用。在此质粒DNA中，有四个限制性核酸内切酶Drai裂解位点存在于 质粒，其中一种残留在三螺旋位点的连接处。形成 Triplex DNA应保护Drai裂解部位，导致消失和外观 具有特定长度的产品，可以通过琼脂糖凝胶电泳很容易检测到。这 拟议工作的结果将是科学界的第一个理解这些工作的努力 新发现的三重结合配体。研究的性质适合培训 以本科生为中心的机构的本科和研究生研究人员。