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

Abasic网站是在细胞DNA中发现的最明显的损伤。因此，它是一种基本的方法，以研究以检测无源位点对DNA损伤的细胞毒性和基因毒性影响，并通过DNA修复良好。在本研究中，一种探测分子（ARP）与无源位点组的特异反应将其合成为DNA中高度敏感和方便的检测的无源位点。一个原氨硫酯涂层的板是用于传统紫外线辐射板以增强DNA绑定的。作为结果，非流动性的时间被缩短到1小时，没有任何信号损失。三标记的DNA数量与盘子的DNA结合在一起是有可能被用于DNA融合的。当ARP中含有DNA的AP站点被处理为解决方案prior涂上保护膜板时，测试的敏感性大大增加了，HeLa RC 355细胞被处理的浓度非常低，这对细胞不起作用。从ARP揭示的细胞中提取的染色体DNA的分析，即每10个低五个碱性位点，D16个D1核otides可以检测到，并演示ARP方法的高敏感性。Furthermore，ARP调查提供了非常有用的检测基础上的DNA N-糖基化酶，在体外和体内进行了异常的检测。与Dojindo Co. Ltd.合作。ARP现在商业上可用，作为DNA损伤检测工具包的一部分，包含ARP、标准的碱性DNA和样本DNA的非流动化平台。

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.