纳米孔单分子检测技术应用于8-羟基鸟嘌呤的检测研究

8-Oxo-guanine is the most frequently occurred DNA damage that leads to mutagenesis in mammals. It is widely accepted as an index for DNA oxidative damage and oxidative stress in cells. Since 8-oxo-guanine plays an important role in the occurrence and development of tumors, it is imperative to develop quick and highly sensitive method for its detection which may provide evidence for cancer early diagnosis and mechanistic studies. The current methods for detecting 8-oxo-guanine include the combination of high performance liquid chromatograph and electrochemical detection, gas chromatograph coupled with mass spectrometry, and enzyme linked immunosorbent assay. We intend to develop a new approach based on nanopore sensing. When the 8-oxo-guanine on a DNA strand is specifically modified with a chemical group, the translocation profile of this DNA through ?-hemolysin nanopre might be drastically altered and this will produce a characteristic event. Statistical analysis of the signature events reveals the position and number of 8-oxo-guanine sites on the DNA strand. The detection does not involve any enzymes or radioactive labeling and requires very little amount of DNA. It makes a nice compliment to the current methods and can be developed as a high-throughput screening tool for 8-oxo-guanine in genomic DNAs.

在DNA氧化损伤中，8-羟基鸟嘌呤是形成频率最高、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，在肿瘤的发生发展过程中具有重要作用。因而对8-羟基鸟嘌呤进行高灵敏、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8-羟基鸟嘌呤主要有高效液相色谱-电化学，气相色谱-质谱，酶联免疫吸附法等，我们拟发展的方法是利用新兴的纳米孔检测技术，使DNA链穿过纳米孔时产生特征信号的方法来检测DNA片段中8-羟基鸟嘌呤的位点和数量，这种方法不需要降解DNA链，不使用任何酶处理或放射性元素标记DNA，且所需DNA量很少，操作简单、快捷，是对现有方法的一个强有力的补充，对各种氧化应激疾病的研究具有重要的意义。

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极为密切。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表达修饰过的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链上氧化损伤造成的8-羟基鸟嘌呤位点提供了一个强有力的工具。