基于哑铃形DNA探针的miRNA抑制剂的设计、构建和功能验证研究

MicroRNAs (miRNAs) are small non-coding RNAs that play important regulatory roles in diverse cellular pathways. Hence, development of their loss-of-function tools is essential for miRNA related fundamental and applicational researches. However, current techniques are found difficult to meet the raising demands on cost and scalability of miRNA inhibition, which significantly limited their application. Our previous researches and other literatures suggested that sealed dumb-bell shaped DNA probes are capable of binding specific miRNA in vitro and in cell lines in a sequence specific manner, which implies their potentials as miRNA inhibitors. In this project, we seek to develop a novel miRNA inhibitor based on the sealed dumb-bell shaped DNA probes building on their advantages on stability, cost and scalability. We intend to customize a novel strategy for probe design and construction, then further characterize their stability and pharmacokinetics. Also, we plan to further explore their effect on miRNA inhibition and their therapeutic applications. In general, in this project, we intend to develop a novel technique for the manipulation of miRNA function and their related pathways. This technique shall provide not only novel tools for miRNA related researches, but also brand-new options for miRNA based therapeutic applications.

miRNA是一类在生命活动中发挥重要调控作用的非编码RNA，开发miRNA抑制剂在基础研究、疾病治疗等领域均具有重要意义。现有miRNA抑制技术在成本和可拓展性等方面存在缺陷，限制了其应用。文献报道和本课题组的前期研究表明，环状哑铃形DNA探针能有效结合目标miRNA，具有开发成为新型miRNA抑制剂的潜力。本项目拟利用哑铃形DNA探针分子结构稳定、易于制作、扩展性强等特点，研究基于哑铃形DNA探针的miRNA抑制剂的设计和模块化合成方法；研究其稳定性、药代动力学特性和生物安全性；在分子、细胞和动物水平上研究哑铃形DNA探针作为miRNA抑制剂的潜力，并探索其在疾病治疗中的应用。本项目旨在开发新型分子探针用于干预miRNA介导的信号转导过程，项目建立的新型miRNA抑制技术不仅能为生命科学基础研究提供新的技术手段，而且有望为基于miRNA抑制剂的临床研究提供新的方法和工具。

miRNA是一类在生命活动中发挥重要调控作用的非编码RNA，开发miRNA抑制剂在基础研究、疾病治疗等应用研究中均具有重大意义。本项目围绕哑铃形DNA探针在miRNA检测和功能干扰方面的应用，探索了此类探针的模块化设计思路、开发了该探针的模块拼接策略，检测了其在不同溶液环境下的稳定性，在分子、细胞水平上开展其功能评估，并开展了拓展性研究。项目研究实现了预定的研究目标，初步建立了基于哑铃形DNA探针的miRNA抑制剂的设计和模块化合成方法，发现环化的哑铃形DNA探针其在核酸外切酶和血清环境中具备良好的稳定性，在分子、细胞等多水平上证实了哑铃形DNA探针作为新型miRNA抑制剂的潜力和效果，开发了基于哑铃形DNA探针的多个miRNA的抑制剂（miRNA-214、622、654、486），发现了miRNA-622、654、486及相关靶基因RNF8、TRIM25的新功能，进一步揭示了肌肉萎缩、肿瘤等疾病的相关发生发展机制。项目建立的新型miRNA抑制技术不仅能为生命科学基础研究提供新的技术手段，而且有望为基于miRNA抑制剂的临床研究提供新的方法和工具。