基于核酸恒温扩增技术的组蛋白修饰超灵敏定量分析方法研究

As an important part of epigenetics, histone modifications play essential roles in many cellular processes including regulation of gene expression. Recently, it has been reported that there is a close relationship between abnormal levels of histone modifications and tumorigenesis, and the histone modifications have attracted wide attentions as novel tumor biomarkers. However, their applications are greatly limited due to the disadvantages of the existed histone modifications assays, such as low detection limit, complicated operations, and long analysis time. Therefore, there is urgent requirement of new methods for histone modifications detection. To achieve this goal, we will develop a series of simple, rapid, and highly sensitive methods for quantitative analysis of histone modifications. The antibody-DNA conjugates are used to transfer the signal of histone modifications to nucleic acid signal, which can be easily amplified. Based on this, the isothermal nucleic acid amplification will be employed to develop novel quantitative histone modifications assays. To further improve the sensitivity, the single molecule detection technology will be introduced to the assay design to develop ultrasensitive platform for histone modifications detection at the single molecule level. The new methods can be applied to clinical cancer detection and the study of tumorigenesis mechanism, providing powerful tools for early diagnosis and treatment of patients.

组蛋白修饰是表观遗传学的重要组成，参与包括基因表达调控在内的一系列重要的细胞生理过程。近年来，研究发现组蛋白修饰水平的异常与肿瘤发生紧密相关，组蛋白修饰作为一种新型肿瘤标志物受到广泛的关注。然而，目前的组蛋白修饰检测方法存在灵敏度不足、操作繁琐、分析时间长等缺点，极大的限制了其在相关领域的发展，亟待开发新型组蛋白修饰分析方法。以此为目标，本项目拟开发一系列简单、快速和高灵敏度的组蛋白修饰定量分析方法。针对蛋白质信号难以放大这一难题，本项目拟利用抗体-DNA共轭物探针，将组蛋白修饰信号转为可方便进行放大的DNA核酸信号。以此为基础，发展基于核酸恒温扩增技术的组蛋白修饰定量分析新方法，并引入单分子检测技术，进一步提高检测方法的灵敏度，构建单分子水平上组蛋白修饰超灵敏检测平台，并将其运用于肿瘤临床早期诊断与发病机理的研究，为患者早诊早治提供有力的工具。

包括组蛋白修饰在内的多种生物分子与疾病发生紧密相关，可作为新型疾病标志物。然而检测方法存在灵敏度不足、操作繁琐、分析时间长等缺点，极大的限制了其在相关领域的发展。针对靶标丰度低、信号难以放大这一难题，本项目开发了多种恒温扩增技术，可对低丰度靶标进行简单有效地扩增，立足点介导的DNA链级联置换、滚环扩增、恒温指数扩增、连接酶扩增、DNA解旋酶依赖扩增、熵驱动DNA纳米机器等。以此为基础，发展基于核酸恒温扩增技术的生物标志物定量分析新方法，实现了包括组蛋白修饰等在内的多种靶标的超灵敏检测，并成功运用于实际生物样品如细胞和临床样品如血液中靶标的准确灵敏分析。本项目发展的新方法新技术将为疾病临床早期诊断提供理论依据和实际应用平台。