新型一锅法滚环扩增技术的建立及其在microRNA检测中的应用

MicroRNAs (miRNAs), a class of endogenous, non-coding small RNAs, play important roles in gene expression regulation. Recent works have also revealed that abnormal expression of miRNAs is closely related to the occurrence and development of cancer and other major diseases in the human body; The development of a simple and fast method to detect microRNAs has important theoretical significance and practical value. Compared with polymerase chain reaction (PCR) method, although the rolling-circle amplification techniques (RCA) have the advantages of high sensitivity and good specificity, there are also the disadvantages of more reaction steps and poor compatibility. This project is aimed at the development of a simple and fast method named one-pot RCA to detect microRNAs. Establishing one-pot RCA. Developing a novel one-pot RCA method based on chemical ligation, also named nonenzymatic autoligation. Studying the effects of different reactive groups at the end of one strand on the abilities of polymerases to replicate or transcribe the sequence and the difference of non-enzymatic method from enzymatic DNA ligation. Establishing an ultrasensitive one-pot RCA method based on target recycling strategy, branched rolling-circle amplification (BRCA) reaction and strand displacement amplification. Investigating the relationship between the expression level of target miRNA and the mutation of p53 gene. Studying the effect of other biomolecules (including drug molecules, etc.) on the miRNA expression in tumor cells.. The effectiveness of the proposed methods will be tested by extended application of these methods to determine in situ the amount of microRNAs in the clinical samples. This research will be helpful for exploring useful diagnostic and prognostic markers of diseases and for further understanding the biological functions of microRNAs as well as early diagnosis of diseases.

MicroRNA（miRNA）作为新的疾病诊断潜在生物标志物，与恶性肿瘤等重大疾病的发生发展密切相关；建立miRNA的快速灵敏检测方法，具有十分重要的理论意义和实用价值。相比于聚合酶链式反应（PCR）检测方法，滚环扩增技术（RCA）虽具有灵敏度高、特异性好、无需热循环仪器和逆转录过程等优势，但也存在反应步骤较多、兼容性差等不足。本课题以建立一锅法RCA技术用于miRNA的快速灵敏检测为目的，分别通过选择性抑制DNA聚合酶的活性、以非蛋白酶连接（如化学连接和DNA酶连接）替代蛋白酶连接、以及利用功能核酸的分子开关功能调控RCA的反应次序，建立基于一锅法RCA的miRNA检测系列新方法；最后将所建立方法用于多miRNA的原位检测及p53 抑癌基因突变与miRNA靶基因表达水平的关系研究。本课题的研究有助于丰富miRNA检测技术，拓展生物医学和临床检测，并在生物和医学研究领域具有重要的意义。

MicroRNA是一类新的疾病诊断潜在生物标志物，其灵敏和快速检测有助于深入了解疾病的发生发展，为疾病的早期诊断提供新技术。本项目以建立一锅法RCA技术用于miRNA的快速便捷检测为目的，通过研究各种连接酶的选择性、聚合酶的外切及聚合活性，建立了快速简便的一锅法RCA新方法；为了提高方法的灵敏度，引入与扩增产物互补的第二引物，建立了超支化扩增的一锅法RCA新方法；结合限制性内切酶，建立了基于靶序列循环的一锅法RCA新方法。同时，结合量子点、银染增强以及生物条形码信号放大技术，结合荧光素探针、纳米金探针以及电化学检测方法，建立了microRNA的快速比色检测系列新方法。本项目的研究将有助于肿瘤标志物的灵敏快速测定，为疾病的早期诊治提供新手段和依据。