RNA干扰过程中靶标mRNA识别机理的单分子成像研究

RNA interference (RNAi) is a unique gene regulation system within living cells. In this process, single strand short interfering RNA (siRNA; 20-30 nt) and Argonaute protein form the core of the ribonucleoprotein complexes - RNA-induced silencing complexes (RISCs). It is well established that the RISCs exert their function in sequence dependent manner and the cleavage mechanism is well understood. However, how the RISC finds its target RNA has remained elusive. We therefore provide mechanistic insights by revealing features of RISC and target RNAs that are crucial to achieve efficiency and specificity in RNA interference by single molecule imaging method. Cy5-RISC and Alexa-555-target RNA were tested to be functional like unlabeled sequence. RISC execute sequence mediates stable association on the immobilized target RNAs and form stable RISC-target complexes, efficient target recognition and cleavage requires only several minutes. We observed that RISC molecule diffused back and forth throughout the cleavage duration multiple times suggesting that RISC may try to cleavage the target multiple times before the final cleavage execution. The single-molecules method developed here opens the door to answering fundamental questions concerning the mechanisms of RISC-target RNA cleavage.

RNA干扰机制自发现起就一直是生命科学领域的研究热点，它揭示了基因表达的调控机制，在生命科学中具有重要作用。在RNA干扰过程中RISC复合体识别靶标mRNA的具体机制还有待进一步研究。单分子成像的技术特点，使它可以提供RNA干扰过程中实时的分子数量变化，以及RISC复合体识别靶标mRNA的具体数据信息，这都是以往分子生物学技术所无法解决的难题，因而能有效地推进目前有关RNA干扰的研究。本研究拟将单分子成像技术应用于RNA干扰的研究，旨在阐明RNA干扰过程中RISC复合体与靶标mRNA识别的具体机理，这对揭示RNA干扰的整体反应过程具有重要意义。本研究的主要目标就是发展超高分辨率的单分子成像技术和单分子操作及测量技术，以便实时观测RISC复合体识别靶标mRNA的具体分子运动过程，在单分子水平上阐明RNA干扰过程中靶标RNA识别的具体过程和反应机理，研究RNA干扰的基因表达和调控机理。