小激活RNA脱靶效应机制及增强其靶向性的策略研究

RNA activation is an important regulation of gene transcription by small RNAs. saRNA is small duplex having similar component but different function compared with that of siRNA. saRNA is expected to be a new type of nucleic acid drugs. saRNA activates gene transcription by targeting the promoter region. The applicants explored the occurrence rules and target recognition mechanism of saRNA in the previous project supported by NSFC. It was for the first time revealed that saRNA is mediated by the “seed region” of antisense-strand and binds directly to promoter DNA to activate gene transcription. The finding is a breakthrough in the mechanism studies of saRNA. The finding that saRNA has a microRNA-like “seed region” drew our attention on the specificity of saRNA target recognition. mRNA chip analysis showed that saRNA could induce off-target effect in target cells, similar with that of siRNA. saRNA showed different mechanism of target recognition from that of siRNA. Therefore, this application will continue to explore the specificity of target recognition and off-target phenomena by using the well-established technology platforms, to explore the possible specific mechanism, to establish a working model for the theory of small RNA regulation. Meantime, we plan to find effective strategies for enhancing target specificity and reducing off-target effect, which will provide principles for saRNA application in vitro and in vivo.

RNA激活是小RNA进行基因转录调控的重要方式。小激活RNA (saRNA) 是与siRNA组成相似而功能不同的双链小分子。saRNA通过靶向启动子区激活目的基因转录。在前一个面上项目中申请人探讨了RNA激活的发生机制，首次揭示了saRNA通过其反义链“种子序列”识别并直接结合靶基因的启动子区DNA，从而介导转录激活过程，这是saRNA机制研究的重要突破。由于saRNA对靶位点的识别具有类似microRNA的依赖种子区域的特征，从而进一步引发了我们对saRNA识靶特异性的关注。前期的mRNA芯片结果提示saRNA与siRNA相似，在靶细胞中存在脱靶效应，saRNA具有与siRNA不同的识靶机制。本申请拟进一步研究saRNA识靶特异性和脱靶现象，探讨其特点和序列识别机制，探索增强saRNA靶向性的有效策略，为小RNA基因调控理论建立新的证据和模型，为saRNA体内外应用提供指导原则。

本项目的主要目标是明确小激活RNA（saRNA）识靶的特异性和脱靶现象，探索序列识别机制及增强saRNA靶向性的有效策略。本项目的主要研究进展如下：1）发现saRNA对多个脱靶基因的序列识别符合“种子序列”调控的规律，非种子序列对于靶点的变异具有较好的耐受性，这是脱靶现象的重要原因；2）saRNA间存在协同激活效果，有助于减少脱靶效应；3）建立了“预激活”的saRNA干预策略，发现saRNA预激活热休克蛋白HSP70可以有效抵御缺氧应激对心肌细胞的损伤，发挥保护作用；4）比较了靶基因的saRNA激活与过表达相比对转录组其它基因表达影响，发现与saRNA的脱靶效应是两种方法存在差异的原因。本项目为saRNA的设计及体内外应用提供了新的实验证据。