单链saRNA加工和抑制效率的研究

项目名称： 单链saRNA加工和抑制效率的研究

项目编号： No.31470781

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 生物科学

项目作者： 吴立刚

作者单位： 中国科学院上海生命科学研究院

项目金额： 80万元

中文摘要： RNAi是由siRNA介导的转录后基因沉默现象，在研究基因功能和疾病治疗中都有很大潜力。但目前用于表达siRNA的shRNA存在较强的脱靶作用和对内源miRNA的竞争抑制，引起副作用。我们研究发现一类双链区较短的siRNA前体-saRNA加工后产生单链siRNA，其脱靶效应和对内源miRNA的影响都显著低于传统的shRNA，有望成为新的RNAi工具。但目前我们对于影响saRNA加工和抑制效率的关键因素及其分子机制还几乎完全未知。因此，我们将研究saRNA加工成熟的分子机制；建立高通量方法筛选高效saRNA，结合生物信息分析，发现影响saRNA加工和对靶基因抑制效率的关键序列和结构特征；并进行动物整体RNAi实验比较saRNA和shRNA的效率和副作用，在转录组水平分析其脱靶效应以及对细胞内源小RNA功能的影响。我们希望通过上述基础研究为saRNA的理性设计和更好的应用打下基础。

中文关键词： RNA干扰；脱靶作用；微小RNA；小干扰RNA；Ago2蛋白

英文摘要： RNA interference (RNAi) is an evolutionarily conserved phenomenon of post-transcriptional gene silencing, triggered by perfect complementary small interfering RNA (siRNA). In mammals, siRNA can be efficiently produced from short hairpin RNA (shRNA), which is transcribed by polymerase III promotors (such as H1, U6). While the classical shRNA is widely used in studying gene function, some hurdle must be overcome before it can be successfully applied to combat human diseases. Firstly, siRNA can target other mRNAs containing sequence that are partially complementary to the guide strand or passenger strand of siRNA, which is known as off-target effect. Secondly, sustained high-level shRNA expression is toxic to cultured cells and adult mouse due to competition with endogenous microRNAs (miRNAs) for the processing factors, such as the nuclear karyopherin exportin-5 and Argonautes. Mammals encode four Argonaute proteins (Ago1-4), which are the core components of RNA-induced silencing complex (RISC). Our previous studies shown that only Ago2 has endonucleolytic activity and has much greater contribution to on-target RNAi than the other non-nucleolytic Ago paralogs, such as Ago1 and Ago3. Recently, we exploited a new siRNA expressing vector which has a short stem and its procession is independent of Dicer but requires Ago2. Only the guide strand of the saRNA remains binding to Ago2 while the passenger strand is discarded after Ago2 cleavage. We named this type of shRNA as Single-stranded Ago2-processed interference RNA (saRNA). The saRNA not only has less off-target effect but also has less competition with endogenous miRNAs comparing to the classically designed shRNA, which provide a valuable alternative tool for RNAi. However the mechanism of saRNA procession and the key factors that determine the procession efficiency and repression activity of saRNA are almost completely unknown. We will develop a high throughput screen method based on FACS and deep sequencing technology to identify the most potent saRNAs from the mixed saRNA library which contains thousands of different saRNAs. By performing bioinformatics analysis on the primary sequence and secondary structure of the most effective saRNAs, the important features that critical for designing efficient saRNA will be identified. We will also identify the ribonuclease responsible for saRNA trimming and investigate the mechanism of how HDV ribozyme cleavage at the 3' end of saRNA can stabilize the saRNA precursor and/or improve its binding to the Ago2. Last, the saRNA expression cassette will be introduced into AAV vector to evaluate the knockdown efficiency and side effects of saRNA in mouse.

英文关键词： RNAi;off-target;microRNA;siRNA;Ago2