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.

项目概要 拟议的研究目标是研究之间的结构-功能关系 三链体DNA和一类新发现的基于类黄酮的三链体特异性结合配体及其用途 所获得的知识可以设计和开发更有效的三链体结合配体作为反基因 疾病治疗的反基因策略中的增强子。该提案可分为三类 部分、结构修饰（合成）、通过生物物理学测定配体-DNA 相互作用 酶活性抑制的方法和研究。综合项目包括改变 连接体长度，去除官能团，并扩展芳香族表面。各结构 修改的目的是解决对结构-功能关系的具体询问。这 合成程序涉及基本有机技术。合成的分子将被用于 通过各种生物物理方法研究它们与多种 DNA 结构的相互作用。稳定化 三链体和双链体 DNA 与配体在不同条件下（pH、离子强度）的分析结果如下： 使用 UC 监测的热变性测定。 DNA的构象变化和 将使用圆二色性测量结合化学计量。选择性结合 DNA 特定构象将通过竞争透析以相对高的通量确定 时尚。配体-三链体 DNA 复合物的转变焓将直接测量 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 （国际贸易委员会）。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。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。含有三链体形成的质粒DNA 将使用网站。在该质粒 DNA 中，四个限制性内切核酸酶 DraI 切割位点存在于 质粒，其中一个残基恰好位于三螺旋位点的连接处。的形成 三链体 DNA 应保护 DraI 切割位点，导致 具有特定长度的产物，可以通过琼脂糖凝胶电泳轻松检测。这 拟议工作的结果将是科学界首次努力了解这些 新发现的三链体特异性结合配体。研究性质适合培训 以本科生为中心的机构的本科生和研究生研究人员。