Chemical Development of Fluorescent Labeling Tags for DNA Damaged Sites

描述(由申请人提供)： 大量研究表明，衰老过程中DNA加合物的积累与癌症的发生有很强的相关性。然而，这些相关性通常并没有导致监测DNA加合物作为监测人类疾病易感性的生物标志物，部分原因是用于检测DNA损伤的生化分析的复杂性。在此，我们建议基于成熟的方法开发新的化学试剂，这些方法将极大地简化对纯化的DNA、组织培养细胞和临床样本中许多DNA加合物的检测。其策略是合成荧光染料衍生物，在使用病变特异性DNA糖基酶切除DNA加合物后，这些衍生物将与DNA骨架反应。我们将用人工合成的方法证明这种方法的可行性，然后用生化方法检测纯化的DNA和哺乳动物细胞中的尿嘧啶和8-氧鸟嘌呤。待开发的染料将考虑膜的通透性，从而允许将这项技术应用于原位测定B细胞淋巴瘤DNA的尿嘧啶含量。该项目的长期目标是开发新的化学和生化工具，以改进现有的方法，在染色体水平上量化和定位人类接触DNA损伤剂的后果。

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.