miRNA和siRNA与AGO蛋白结合后形成RISC复合物行使调控功能，因此miRNA和siRNA对靶基因的调控效率和特异性，不仅取决于miRNA和siRNA与靶位点序列碱基配对的结构和稳定性，也依赖于AGO蛋白的特性。哺乳动物编码四个AGO蛋白（AGO1-4），其中只有AGO2具有核酸内切酶活性，并且各个AGO成员间的相对表达量在各个组织中有较大差异。但不同的AGO蛋白识别靶位点的特征以及功能上是否存在差异，目前都尚不清楚。我们近期研发了基于共价交联的SpyCLIP技术，为研究蛋白质-RNA相互作用提供了简便、精确的工具。我们将运用SpyCLIP技术，发现AGO2内切核酸酶活性依赖型的靶基因，揭示不同AGO蛋白特异性的miRNA靶基因及其功能；鉴定siRNA和saiRNA的脱靶位点，发现脱靶效应的规律，为理解miRNA的靶基因识别规则和提高RNAi的专一性提供理论基础。

microRNAs (miRNAs) can repress thousands of genes in in animal cells by annealing to mRNAs to which they are imperfectly complementary. RNA interference (RNAi) is an evolutionarily conserved phenomenon of post-transcriptional gene silencing, triggered by perfect complementary small interfering RNA (siRNA). 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. The function of both miRNA and siRNA depends on the Argonaute proteins they are associated with. Mammals encode four AGO 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. However, whether the different AGO-miRNA complex can target and regulate different set of genes in the cells remains elusive. Recently, we invented a SpyCLIP method that employs a covalent linkage formed between the RBP-fused SpyTag and SpyCatcher, which can withstand the harshest washing conditions for removing nonspecific interactions, providing a robust method for both routine characterization and high-throughput studies of protein-RNA interactions. In this project, we will use AGO-SpyCLIP to investigate the miRNA targets associated with different AGO proteins in the cells and their biological function, as well as the off-target effect caused by siRNA and saiRNA, which may lead to novel strategies for designing better siRNAs with less nonspecific effect for gene therapy.