用于规模性研究蛋白质-RNA相互作用的新型CLIP方法研发

Growing numbers of proteins are found to be associated with RNAs and play critical roles in both normal and pathological processes. UV cross-linking and immunoprecipitation (CLIP) coupled with high-throughput sequencing is the most state-of-the-art technology to characterize protein-RNA interactions, yet only a small portion of RNA-binding proteins (RBPs) have been thoroughly studied due to its requirement for RBP-specific antibodies and complicated experimental procedure. In this proposal, we describe a non-antibody-dependent SpyCLIP method employing a covalent link formed between the RBP-fused SpyTag and the bead-coupled SpyCatcher, which can withstand the harshest washing conditions and circumvent the RNA labelling and gel purification steps needed for all existing CLIP methods. Moreover, SpyCLIP is performed on beads and readily amenable to automation, which provides an efficient and easy-to-use method for both routine and high-throughput characterization of protein-RNA interactions without scarifying quality. We believe this technique would liberate scientists from the frustration of trying to get high quality protein-RNA interaction information and accelerate our understanding of the fascinating and complex mechanisms of RBPs.

目前已知的RNA结合蛋白已经超过一千种，许多过去被认为不结合RNA的蛋白质近期亦被发现具有RNA结合能力，所结合的RNA对调控这些蛋白质机器的活性发挥了重要作用。CLIP是研究体内蛋白质-RNA相互作用最为先进的方法之一。该方法历经改进，产生了多种变体，但现有CLIP版本均依赖于高特异性的抗体，实验步骤繁杂，大数据的可重复性差，无法规模化应用。因此，研发简洁高效且适合高通量应用的新方法是系统性理解蛋白质-RNA相互作用的关键和当务之急。本项目采用一种全新的基于共价交联的纯化策略，可以极大地提高所获蛋白质-RNA复合物的纯度。同时对CLIP建库的流程进行创新设计，避免传统CLIP方法中多步限制规模化操作和大量损耗起始材料的效率限制性步骤。这些核心改进不仅可以摆脱现有CLIP方法对抗体的依赖，提高数据的信噪比和可重复性，并大幅降低了操作的难度和时长，使得CLIP方法能够适应高通量研究的需求。