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Development of novel probe molecules for specific detection of abasic sites in DNA

开发用于特异性检测 DNA 脱碱基位点的新型探针分子

基本信息

批准号：
09558070
负责人：
IDE Hiroshi
金额：
$ 5.18万
依托单位：
Hiroshima University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

项目摘要

Abasic sites are the most ubiquitous damage found in cellular DNA. Therefore, it is essential to develop a method to detect abasic sites to study cytotoxic and genotoxic effects of DNA damage as well as DNA repair.In this study, a probe molecule (ARP) specifically reacting with the aldehyde group of abasic sites was synthesized for highly sensitive and convenient detection of abasic sites formed in DNA.For immobilization of DNA, a protamine sulfate-coated plate was used instead of the conventional UV-irradiated plate to enhance DNA binding. As the result, the time for immobilization was shortened to 1h without any loss of signals. The amount of tritium-labeled DNA bound to the plate was proportional to the DNA concentration employed. When DNA containing AP sites was treated with ARP in solution prior to coating the protamine-plate, the sensitivity of the assay was greatly increased.HeLa RC355 cells were treated by a very low concentration of an alkylating agent (MMS) that does not affect cell survival. The assay of chromosomal DNA extracted from the cell with ARP revealed that as low as five abasic sites per 10ィイD16ィエD1 nucleotides could be detected, demonstrating a high sensitivity of the ARP method. Furthermore, the ARP assay proved to be very useful for detection of abasic sites resulting from excision of damaged base by DNA N-glycosylases in vitro and in vivo.In collaboration with Dojindo Co. Ltd., ARP is now commercially available as a reagent or a part of the DNA damage assay kit containing ARP, standard abasic DNA, and plates for immobilization of sample DNA.

非碱性位点是细胞DNA中最普遍的损伤。本研究合成了一种能特异性地与DNA脱碱基位点的醛基反应的探针分子（阿普），用于检测DNA中形成的脱碱基位点。使用硫酸鱼精蛋白包被的板代替常规的UV照射的板以增强DNA结合。结果，固定化时间缩短到1h，没有任何信号损失。与板结合的氚标记DNA的量与所用DNA浓度成比例。当含有AP位点的DNA在包被鱼精蛋白板之前在溶液中用阿普处理时，检测的灵敏度大大增加。从细胞中提取的染色体DNA与阿普的分析表明，每10个碱基D16碱基D1核苷酸可以检测到低至5个脱碱基位点，证明了阿普方法的高灵敏度。此外，阿普分析被证明对于在体外和体内检测由DNA N-糖基化酶切除受损碱基而产生的脱碱基位点非常有用。阿普现在可作为试剂或DNA损伤测定试剂盒的一部分商购获得，所述试剂盒含有阿普、标准无碱基DNA和用于固定样品DNA的板。