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

*这项研究计划旨在推进研究DNA损伤和突变的分析技术。拟议的研究集中在：(1)开发用于测量特定基因位置上的DNA损伤的分析技术；(2)成像活细胞中的特定DNA突变；以及(3)将新技术应用于实时监测细胞中的分子过程。人类DNA中特定部位的损伤已知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损伤检测方法变得至关重要。这项研究的第一个目标是开发一种新的分析技术，可以定位靶基因上的特定损伤，同时定量损伤的丰度。这项技术使用了两个设计合理的分子探针，一个与DNA损伤结合，另一个与特定基因结合。这两个探针与同一个DNA分子结合产生了一个新的可检测的DNA组件。这项超灵敏技术将应用于检测环境和临床因素造成的特定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新的方法，对活细胞中的DNA突变进行成像。这种新方法利用了一种名为CRISPR的强大基因组工程技术。这项技术使用一组分子来定位基因组中的目标位置，在目标位置对DNA进行切割，然后对切割的部分进行编辑和修复，在目标位置完成基因组编辑。在这项研究中，CRISPR独特的定位和结合基因组特定位置的能力将被用来将一个特殊设计的荧光探针输送到目标位置。将该探针与DNA中的特定突变结合将打开荧光。监测细胞中产生的荧光将使突变的实时成像成为可能。*第三个目标是将新技术的应用扩展到研究分子相互作用和可视化细胞内的化学过程。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.