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

项目摘要 拟议的研究目标是获得一种先进的凝胶成像系统，以提高 正在进行的研究三链DNA和一类 新发现的类黄酮基三链体特异性结合配体。配体介导的 三链体的形成可用于在疾病的抗基因策略中设计有效的抗基因增强子 治疗该凝胶成像系统将用于三组实验。1)的质粒DNA 将构建含有四个限制性内切酶DraI切割位点的质粒。其中一个Drai 切割位点位于三链体形成区。配体介导的三链体形成将保护 这个切割位点来自DraI消化。切割的DNA片段可以通过琼脂糖凝胶分离 电泳并通过荧光在新的凝胶成像系统上观察。基于DNA 从DraI消化获得的片段，我们可以确定配体介导的三链体的效率 形成抑制酶活性。将获得高质量的凝胶图像。2)荧光 将合成探针标记的形成三链体的DNA寡核苷酸。双链体的嘌呤链 DNA用绿色探针标记，三链体形成寡核苷酸用红色探针标记。 将使用聚丙烯酰胺凝胶电泳分离形成的DNA。配体介导的 三链体形成将导致代表双链体和三链体形成的不同颜色方案 DNA（双链体DNA：绿色条带;三链体DNA：黄色条带;无双链体或三链体DNA形成： 分离的红色和绿色带）。这种颜色方案可以很容易地观察到使用多重检测 新的凝胶成像系统3)含有红色荧光蛋白（mcherry）的质粒DNA将 被建造。mcherry序列的上游含有三链体形成位点。配体- 介导的三链体形成将减少活细胞中红色荧光蛋白的合成（E. coli）。的 活细胞（体内）中的荧光可以使用新凝胶上的基于平板的成像来可视化 成像系统先进的凝胶成像系统的收购使我们能够进行高质量的 凝胶成像实验提出了在父母奖与24/7访问，并扩大我们的研究 能够进行多重成像和体内成像。此外，它还介绍了最先进的 成像技术，我们的本科生和研究生，以加强他们的经验学习 在太平洋的经验，并改善我们的本科为中心的研究基础设施 机构。

Project Summary The proposed research objective is to acquire an advanced gel imaging system to enhance the ongoing research on studying the structure-function relationship between triplex DNA and a class of newly discovered flavonoid-based triplex-specific binding ligands. Knowledge of ligand-mediated triplex formation can be used to design potent antigene enhancers in the antigene strategy for disease treatment. This gel imaging system will be used in three sets of experiments. 1) A plasmid DNA containing four restriction endonuclease DraI cleavage sites will be constructed. One of the DraI cleavage sites resides in a triplex-forming region. The ligand-mediated triplex formation will protect this cleavage site from DraI digestion. The cleaved DNA fragments can be separated by agarose gel electrophoresis and observed on the new gel imaging system via fluorescence. Based on the DNA fragments obtained from DraI digestion, we can determine the efficiency of ligand-mediated triplex formation on inhibition of enzymatic activities. High-quality gel images will be obtained. 2) Fluorescent probe labeled triplex-forming DNA oligonucleotides will be synthesized. The purine strand of duplex DNA is labeled with a green probe, and the triplex-forming oligonucleotide is labeled with a red probe. The formed DNA will be separated using polyacrylamide gel electrophoresis. The ligand-mediated triplex formation will result in different color schemes representing the formation of duplex and triplex DNA (duplex DNA: a green band; triplex DNA: a yellow band; no duplex or triplex DNA formation: separated red and green bands). This color scheme can be readily observed using multiplex detection on the new gel imaging system. 3) A plasmid DNA containing a red fluorescent protein (mcherry) will be constructed. The upstream of the mcherry sequence contains a triplex-forming site. The ligand- mediated triplex formation will reduce the synthesis of red fluorescent protein in live cells (E. coli). The fluorescence in live cells (in vivo) can be visualized using the plated-based imaging on the new gel imaging system. The acquisition of an advanced gel imaging system allows us to conduct high-quality gel imaging experiments proposed in the parent award with 24/7 access and to expand our research capability to perform multiplex imaging and in vivo imaging. In addition, it introduces state-of-the-art imaging technology to our undergraduate and graduate students to enhance their experiential learning experience at Pacific and improve the research infrastructure at our undergraduate-centered institution.