单链saRNA加工和抑制效率的研究

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.

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。

RNAi 是由siRNA介导的特异性降解具有互补序列的RNA，从而在转录后水平沉默靶基因表达的现象。目前已被广泛应用于基因功能研究，并能作为小核酸药物直接用于疾病治疗。但RNAi也存在毒副作用，主要包括非特异性抑制靶基因以外其它基因的脱靶作用，以及表达shRNA生成siRNA时对细胞内源miRNA的竞争抑制作用。因此，亟待设计更好的RNAi载体在高效沉默靶基因的同时降低其毒副作用。我们研发了一种较传统shRNA更为高效、安全的单链siRNA表达载体：核酶增强型saiRNA。saiRNA的茎环结构具有较短的双链区，由Ago2蛋白直接在双链区3’臂进行切割，并进一步在细胞内被加工生成长度为24-27nt的单链siRNA。通过在saiRNA的3’末端融合HDV核酶，利用核酶的高效自切割活性准确地在saiRNA的3’端产生两个碱基的悬垂，大大提高了saiRNA前体与Ago2蛋白的结合效率，从而显著增强了其对靶基因的沉默效率。新型的saiRNA载体仅产生一条单链siRNA，并特异性与Ago2结合，相较于传统的shRNA具有更小的脱靶作用；在同样的沉默效率下，saiRNA产生的成熟siRNA在细胞内的积累量远低于shRNA，减少了对细胞内源miRNA加工和功能的竞争作用。因此，新型的saiRNA作为一种高效、低副作用的RNAi载体，为科研和疾病治疗提供了新工具。另外，我们还基于spyTag和spyCatcher共价交联作用研发了新型CLIP技术—spyCLIP，利用该系统可以经受高强度洗脱的特性对待研究的RBP进行纯化，避免了传统CLIP技术中对特异性抗体的需求，高效去除非特异性结合杂质，并可避免多个实验限速及低效率步骤，大大简化了实验并提高了数据的信噪比。我们通过Ago2-spyCLIP可以更为精准的判定RNAi的脱靶效应，为进一步优化saiRNA设计方案提供了重要的工具。