cRGD修饰的光敏感环状siRNAs的合成及其光调控基因表达研究

Controlling the release of small interfering RNAs (siRNAs) in spatiotemporal and concentration dependent mode is very important for their gene function study. The application of photolabile groups provides a convenient method for spatiotemporal regulation of siRNAs expression. Several unwanted effects of siRNAs such as enzymatic degradation, nonspecific delivery and off-target effects inhibit the photoregulation of RNAi. Based on our previous studies, we design and synthesize a novel type of photolabile siRNAs with abilities of specific delivery to target cells, reducing off-target effects and enhancing long-term gene silencing, that is caged circular siRNAs with cRGD functionalization (caged circular antisense RNAs/sense RNAs with 5' end cRGD modification, caged antisense RNAs with 5' end cRGD modification/circular sense RNAs). We will investigate the photoinduced release of active siRNA, the affinity of the cRGD receptor with cRGD attached on siRNAs, serum stability, off-target effects as well as the gene silencing ability before and after light activation of caged siRNAs. Further studies about the mechanism of siRNA delivery and activation for these caged circular siRNAs with cRGD modification will also be demonstrated. It will pay the way for spatiotemporal and concentration photoregulation of specific gene expression and circumvent off-target gene silencing by the sense strand.

在时间、空间、浓度上调控细胞中siRNAs的递送和释放对RNAi的基因功能研究具有重要的意义。光敏感基团的应用为时空调控siRNAs活性提供了便捷方法。但是siRNAs自身的核酶不稳定、缺乏靶向性和脱靶效应降低RNAi的光控效果。本项目在前期工作的基础上，设计合成一种新型靶向性、长效性、降低脱靶效应的光敏感siRNAs：cRGD修饰的光敏感环状siRNAs（光敏感环状反义链/5'末端cRGD修饰的正义链，5'末端修饰cRGD的光敏感反义链/环状正义链）。研究其光控释放能力、受体结合能力、稳定性、脱靶效应，以及在受体高表达细胞中对外源和内源基因表达沉默活性的抑制和光激活后siRNAs沉默活性的恢复，阐明在光激发前后对其在细胞内递送、分布和活性光调控机制，为在时间、空间和浓度上调控光敏感siRNAs对特定基因表达及消除正义链导致的siRNA脱靶效应奠定基础。

在时间、空间、浓度上调控细胞中基因表达对基因功能研究具有重要意义。本项目设计、合成靶向基团修饰光敏感环状siRNAs[光敏感环状反义链/3'末端GalNAc修饰的正义链（CpAG/Gal-SG），5'末端修饰cRGD的光敏感反义链/环状正义链(RpAG/CSG)]来调控生物活性，实现靶向基因表达的光调控。首先合成一种光敏感的1-(2-硝基苯)乙醇的亚磷酰胺单体和末端具有NHS活化羧基亚磷酰胺单体，并将其通过DNA/RNA固相合成仪依次插入到RNA序列5’末端，与氨基修饰的cRGD肽作用形成酰胺键得到cRGD修饰的光敏感RNA序列。环状RNAs通过5’末端磷酸基团与3’末端羟基在T4 RNA liagse 1作用下形成磷酸二酯键得到。考察RpAG/CSG和CpAG/Gal-SG在细胞内siRNA活性的抑制和光照恢复能力。光照之前，环状siRNAs靶向基团cRGD和GalNAc的修饰比未修饰的siRNAs能更好封闭siRNA活性，降低脱靶效应，但环状结构使RpAG/CSG的光恢复能力减弱，而光照恢复成线性结构的CpAG/Gal-SG的基因沉默活性更佳。此外，反义链cRGD距离5’末端的空间距离对siRNAs活性起到关键作用，当连接臂过长时，不能有效封闭基因沉默活性。这部分研究对于理解环状siRNAs的结构修饰和光调控基因表达具有重要的意义。