Ultra-sensitive assays for DNA damage, mutation, and DNA-protein interaction

这个研究项目旨在推进研究DNA损伤和突变的分析技术。建议的研究重点是：(1)开发测量特定基因位置DNA损伤的分析技术；(2)成像活细胞中的特异性DNA突变；(3)将新技术应用于细胞分子过程的实时监测。******已知人类DNA中特定位点的损伤会导致各种不良后果。DNA损伤的关键作用使得开发高灵敏度和特异性的DNA损伤检测方法至关重要。本研究的第一个目标是开发一种新的分析技术，该技术将定位目标基因上的特定损伤，同时量化损伤的丰度。该技术使用两种合理设计的分子探针，一种结合DNA损伤，另一种结合特定基因。两个探针结合到同一个DNA分子上，产生一个新的可检测的DNA组装体。这种超灵敏技术将用于检测由环境和临床因素引起的特定DNA损伤，如紫外线、癌症治疗药物和环境污染物。******第二个目标是建立一种在活细胞中成像DNA突变的新方法。这种新方法利用了一种强大的基因组工程技术——CRISPR。该技术利用一组分子在基因组中定位目标位点，对目标位点的DNA进行切割，然后对切割进行编辑和修复，完成目标位点的基因组编辑。在本研究中，CRISPR定位和结合基因组特定位点的独特能力将被用来将一种特殊设计的荧光探针传递到目标位点。将探针与DNA中的特定突变结合将开启荧光。监测细胞中产生的荧光将使突变的实时成像成为可能。******第三个目标是扩展新技术的应用，以研究分子相互作用和可视化细胞中的化学过程。所提出的分析技术和方法的概念和策略也适用于许多其他具有生物化学、环境和临床重要性的化合物。

***This research program aims to advance analytical technology for studying DNA damage and mutation. The proposed research focuses on: (1) developing analytical techniques for measuring DNA damage on specific gene locations; (2) imaging specific DNA mutations in live cells; and (3) applying the new techniques to real-time monitoring of molecular processes in the cells. ******Damage at particular sites in human DNA is known to lead to various adverse outcomes. The critical role of DNA damage makes it essential to develop highly sensitive and specific assays for DNA damage. The first objective of this research is to develop a new analytical technology that will locate the specific damage on the target gene and quantify the abundance of the damage at the same time. This technology uses two rationally designed molecular probes, one binding to the DNA damage and the other binding to the specific gene. Binding of both probes to the same DNA molecule produces a new detectable DNA assembly. The ultra-sensitive technique will be applied to the detection of specific DNA damage caused by environmental and clinical agents, such as ultraviolet light, cancer treatment drugs, and environmental contaminants.******The second objective is to establish a new method for imaging DNA mutations in live cells. The new method takes advantage of a powerful genome engineering technology, called CRISPR. This technology uses a set of molecules to locate the target site in the genome, make cuts to the DNA at the target site, and then edit and repair the cuts, accomplishing genome editing at the target site. In this research, the unique ability of CRISPR to locate and bind to specific sites of the genome will be used to deliver a specially designed fluorescence probe to the target site. Binding of this probe to the specific mutations in the DNA will turn on the fluorescence. Monitoring of the fluorescence generated in the cells will enable real-time imaging of the mutations. ******The third objective is to extend the applications of the new techniques to studying molecular interactions and visualizing chemical processes in the cells. The concept and strategy of the proposed analytical technologies and methods are also applicable to many other compounds of biochemical, environmental, and clinical importance.