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

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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.