microRNA灵敏检测及其与环境污染物相关性研究

Mature microRNAs are a class of evolutionally conserved, single-stranded, small 20-26 nucleotide(nt) long noncoding RNA. Mounting evidence has implicated microRNA (miRNA) expression profiles are effective biomarkers for a variety of diseases, including cancer, heart, obesity, and mental illness because aberrant miRNA expression profiles have been linked to the development of several disease types. Moreover, Organisms are exposed to pollutants cause the expression of relevant miRNA changes, which lead to its target gene expression changes. It is meaningful and significant to detect microRNA for diseases diagnostic and genetic toxicology research of pollutants. However, conventional miRNA detection technologies such as microarray analysis and quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR), while can serve as research tools, require expensive reagents, instrumentation, and multiple time-consuming steps. The aim of project is to develop a new electrochemiluminescence sensor based on DNA charge transfer, and detect microRNA with high specificity, high sensitivity using electrochemiluminescence and electrochemistry techniques. The correlation between miRNA and pollutants would be studied. A set of detection mechanism with high sensitivity and specificity and the correlation curve would be presented. The project results would provide theoretical and technical supports for miRNA detection development, genetic toxicology of pollutants, and DNA charge transfer applications.

MicroRNA是一类长度为20～26 个核苷酸的非编码内源性单链小分子RNA。当生物暴露于环境污染物时会引起相关microRNA表达发生变化，进而导致其靶基因表达发生改变,且MicroRNA的表达水平与许多疾病密切相关。所以，microRNA的定量检测对相关疾病的诊断与治疗以及环境污染物的基因毒理研究具有重要意义。然而通常的microRNA检测技术如：逆转录聚合酶链式反应等存在试剂及仪器昂贵、耗时等缺点。本项目拟以DNA电荷传递对碱基堆积结构高度灵敏的依赖性为理论依据，研制高效检测microRNA的电化学发光传感器,以电化学及电化学发光为主要技术手段系统研究microRNA检测平台的影响因素以及microRNA与环境污染物的相关性，提出一套完整的灵敏、特异检测机制，构建相关性曲线。为提高microRNA检测水平、揭示环境污染物基因毒理以及进一步扩展DNA电荷传递的应用提供理论和技术支持。

MicroRNAs是一类含有19-23个碱基的非编码单链小分子RNA，通过碱基互补与mRNA序列的3′UTR区或编码区不同程度结合从而调控靶基因的表达，在动植物的发育、细胞生长分化和凋亡、代谢等生命活动过程中发挥重要的调控作用。有关miRNA研究的基础是对其高灵敏和选择性的测定。如何切实增强电化学发光信号实现microRNA的灵敏测定是该项目的主要研究内容。首先基于核酸放大机制我们设计了双联特异性酶和杂交链式反应的双重放大方法实现了microRNA的超灵敏检测；其次在分析电化学发光机理的基础上设计了共反应剂原位产生进一步增强了电化学发光信号；我们还开发了新的电化学发光淬灭体系-甲基蓝淬灭三联吡啶钌。并在研究中发现优化DNA探针的固载方法和状态对提高检测的灵敏度具有重要意义。设计并应用了三脚架结构的ＤＮＡ探针和点击化学组装方法获得了高杂交效率的均一化传感界面。细胞水平的MicroRNAs测定对研究环境污染物的毒理提供了新的技术。