Chemical Development of Fluorescent Labeling Tags for DNA Damaged Sites

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DESCRIPTION (provided by applicant): Numerous studies have shown a strong correlation between accumulations of DNA adducts during aging and carcinogenesis. However, these correlations have not generally resulted in monitoring of DNA adducts as biomarkers for monitoring susceptibility to human diseases, in part, because of the complexities of biochemical assays for the detection of lesions in DNA. Herein, we propose to develop new chemical reagents based on well established methodologies that should greatly simplify detection of many DNA adducts in purified DNA, tissue culture cells and clinical samples. The strategy is to synthesize fluorescent dye derivatives that will react with DNA backbone following excision of DNA adducts using lesion-specific DNA glycosylases. We will demonstrate the feasibility of this approach by using synthetic, followed by biochemical approaches for detecting uracils and 8- oxoguanines in purified DNA and mammalian cells. The dyes to be developed will be considered for membrane permeability allowing the application of this technology to determining uracil content of B cell lymphoma DNA in situ. The long term goal of this project is to develop new chemical and biochemical tools to improve upon existing methodologies, to quantify and localize, at chromosome level, consequences of human exposure to DNA-damaging agents.