基于模板识别的microRNA检测平台研究

MicroRNA (miRNA) is a class of endogenous non-coding length of single-stranded RNA of about 20 nucleotides, widely present in various body fluids and tissues, it has become an important biomarkers for cancer and other diseases and therapeutic targets. My group has been developing novel chemiluminescence (CL) technologies for many years and already published 11 research papers in the prestigious journal of "Analytical Chemistry". In 2011, we first presented a sequence-specific detection method of short-length DNA via template-dependent surface-hybridization events and won the 2012 NSFC funding. The goal of this project is to further improve and extend previous techniques demonstrated in the concluding report, in order to avoid the following drawbacks including complicated design of reporter probe, low sensitivity for low levels of miRNA in the easily accessible body fluid samples and difficult discrimination for those closely related sequences from the miRNA family members. In sharp contrast to previous methods, we will focus on the development of new template recognition-based detection technology to improve the detection sensitivity and selectivity and operability, in order to meet the analytical needs of a wide range of normal and patient body fluid samples. We believe such advancements may be possibly used for early disease diagnosis and prognosis, and also provide important information about the roles of miRNAs in normal cell functions and disease.

MicroRNA（miRNA）是一类长度在20个核苷酸左右的内源性非编码单链RNA，广泛存在于人各种体液及组织中，已成为癌症等疾病的重要标志物和治疗靶点。申请人研究小组多年来一直致力于分析新技术的研究，研究成果已11次发表在国际著名分析化学杂志《Analytical Chemistry》上，2011年首次提出并报道了基于模板识别的短链DNA化学发光检测技术，获得了2012年面上项目资助。本课题即是上述结题课题的进一步提升和发展，拟针对目前模板识别平台中报告探针设计繁琐、易获取体液样本miRNA含量很低以及家族序列单碱基分辨困难等核心问题，突破以往思维，发展新型的miRNA模板识别检测技术，提高检测灵敏度和选择性以及可操作性，满足大范围正常人和病人体液样本中miRNA的分析需要，以期应用于癌症等疾病的早期诊断和预后疗效监测，进一步了解miRNA的确切生物生理功能及其在疾病发生中所扮演的角色。

MicroRNA（miRNA）广泛存在于各种体液及组织中，已成为癌症等疾病的重要标志物和治疗靶点。按照原定计划，本课题针对报告探针设计繁琐、易获取体液样本miRNA含量很低以及家族序列单碱基分辨困难等核心问题展开了一系列研究，主要包括：1）采用切刻内切酶、滚环、杂交链式和催化发夹组装恒温扩增反应，构建了多种基于恒温扩增的miRNA灵敏检测技术；2）偶合多种放大技术如微球和杂交链式反应、DSN和TdT的双重信号放大等模式，实现了miRNA的双重放大检测。上述方法中采用了化学发光、荧光和质谱作为miRNA的检测手段，研究发现：1）化学发光和质谱法更适合于单组分miRNA的体外检测，流式荧光平台技术很适合多组分miRNA的灵敏检测；2）选择合适的滚环扩增连接酶和RNase One核糖核酸酶，能部分消除miRNA的错配杂交；3）主动引入单碱基突变位点至padlock设计中，能够进一步提高miRNA家族序列的错配识别能力。在以上工作的基础上，进一步拓宽发展了多种基于核酸序列设计的端粒酶和转录因子等检测技术，并为下一步细胞和活体动物水平上miRNA的检测，构建了多种小分子生物发光探针；应邀撰写化学发光检测技术综述一篇。基于本课题，已发表研究论文16篇，协助培养博士研究生4名，硕士研究生3名。