10-23型脱氧核酶对mRNA表达的双重抑制作用

10-23 DNA enzyme has twofold cleaving effects on target mRNA

目的:10-23型脱氧核酶是脱氧核糖核酸酶中的一种,能主动在RNA分子嘌呤-嘧啶结合点切割所有RNA分子,通过比较反义寡核苷酸的作用,初步研究10-23型脱氧核酶对基因表达抑制作用的发生机制。方法:采用体外转录GFP mRNA与脱氧核酶反应,以及GFP表达质粒与脱氧核酶共转染HeLa细胞,观察脱氧核酶对GFP的抑制作用。结果:体外和胞内实验均显示据靶点设计的10-23型脱氧核酶对绿色荧光蛋白mRNA具有切割作用。当10-23型脱氧核酶的两翼结合臂各具有连续8碱基互补而处在两翼结合臂和酶解核心连接处的2碱基错配时,尽管不能切割mRNA但是在胞内仍然有表达抑制作用;而当两翼结合臂具有4互补碱基紧接1错配碱基和4互补碱基时,10-23型脱氧核酶体外和胞内均丧失作用。结论:10-23型脱氧核酶对基因较强的表达抑制作用来自结合臂的反义寡核苷酸依赖的RNase H降解,和本身对mRNA直接切割作用的叠加,即10-23型脱氧核酶在细胞内具有对基因识别切割和诱导RNase H进行再切割的双重作用。

Objective： To study preliminarily the mechanism of gene knock down by 10-23 DNA enzyme can cleave almost any target RNA that contains a purine-pyrimidine（R-Y） junction. Methods： In vitro, 10-23 DNA enzyme reacted on the transcripted GFP mRNA. In vivo, the inhibiting effect of 10-23 DNA enzyme on the GFP was observed by co-transfecting 10-23 DNA enzyme and GFP expressing plasmid into HeLa cells. Results： The knock down effect of 10-23 DNA enzymes designed according to screened sites on GFP was obvious in vitro and in vivo. A 10-23 DNA enzyme that had arms consisting of continuously matching 8nt bases and 2 bases mutations at the arm-core joints, lost cleavage effect on the mRNA in vitro, but still showed knock down effect on GFP in vivo. When each ann had 4 matching bases plus 1 mismatching base and 4 matching bases, the 10-23 DNA enzyme had no cleavaging effect in vitro and no knock down effect in vivo. Conclusion：It was demonstrated that the knock down of the GFP gene by 10-23 DNA enzymes resulted from the direct cleavage together with RNase H dependent splitting. It is suggested that 10-23 DNA enzyme has hydrolyzing effect and additional cleaving effect by recruiting RNase H on mRNA. In other words, the 10-23 DNA enzyme has twofold effect on the mRNA that they can recognize and cleave the mRNA and can cleave the mRNA through the way of RNase H.