DNA-致癌物加合物的纳米孔检测研究

Cancer has been the number one killer disease in the new century. About 90% of the tumors are caused by synthesized compounds-carcinogen. Mechanistic studies have shown that carcinogens enter cells and react with DNA to form adducts. Those adducts may undergo further reactions leading to DNA chain breaking or base modification. If organisms fail to repair those DNA damages, mutations may occur. Therefore, rapid and sensitive detection of DNA-carcinogen adducts is very important for early diagnosis of cancer and mechanistic studies of carcinogenesis. Currently, there are mainly four types of methods for detecting DNA adducts. They are 32P-post-labelling, quantitative immunoanalysis, fluorescence measurement and HPLC-MS etc. The method we aim to develop relies on the novel nanopore technology. We will fabricate a nanopore system based on single-walled carbon nanotubes. When single-stranded DNA traverses through the nanopore, it will cause ionic current changes at picoampere level. If the DNA has carcinogen adducts on it, the current will alter according to the positions and numbers of the reacted sites. This strategy deals with single-stranded DNA rather than degraded nucleotides and does not rely on any enzymes or antibodies. It requires only microgram scale samples and the detection can be accomplished within one hour. It is a nice complement to the current approaches of detecting DNA-carcinogen adducts and potentially useful for early diagnosis of cancer.

恶性肿瘤（癌症）已经成为新世纪威胁人类健康的第一杀手。人类90%的肿瘤是由人工合成的化合物引起的，肿瘤形成的机理研究表明，化学致癌物进入到生物体内，与细胞内的DNA发生相互作用，生成DNA加合物，导致链断裂或碱基被修饰，从而导致遗传信息的改变而诱发变异。因而对DNA与致癌物生成的加合物进行高灵敏、快速检测可以为致癌机理研究和癌症早期诊断提供科学依据。目前检测DNA加合物主要有32P后标记法，免疫分析法，荧光法，色谱-质谱法等。我们拟发展的方法是利用新兴的纳米孔检测技术，构建基于碳纳米管的纳米孔传感体系，并利用DNA链穿过纳米孔时引起的皮安级电流信号的变化来检测DNA链上致癌物的加合位点和数量。这种方法不需要降解DNA链，不使用任何酶或抗体处理DNA，所需DNA量很少（小于1微克），操作简单、快捷，是对现有方法的一个强有力的补充，对促进致癌物的致癌机理研究和肿瘤的早期诊断具有重要的意义。

在DNA 氧化损伤中，8-羟基鸟嘌呤（OG）是形成频率最高、致突变能力最强的产物，被公认为DNA 氧化损伤和细胞内氧化应激的指示剂，与肿瘤的发生和发展关系也最为密切。鉴于8-羟基鸟嘌呤在DNA损伤研究中的重要作用，我们采用对8-羟基鸟嘌呤进行特异性修饰的策略来检测DNA链中的鸟嘌呤氧化位点。当金刚烷-瓜环络合物修饰的DNA片段穿过溶血素纳米孔时，会产生两种类似但稍有区别的特征电流信号。经过对DNA穿越纳米孔的机制的研究，我们发现这两种信号均可以用来研究修饰过的DNA穿越纳米孔的过程，且可以合并统计分析。利用已知比例的OG-DNA与普通DNA混合，建立了特征信号频率与OG-DNA百分含量的标准工作曲线。该方法可以测量出DNA混合物中OG-DNA的含量约1%的变化，为快速检测DNA氧化损伤造成的8-羟基鸟嘌呤位点提供了一个强有力的工具。