摘要
10-23DNA酶是能主动切割mRNA的一类反义寡核苷酸.利用10-23DNA酶的直接切割作用验证mRNA结构靶点的有效性.对筛选的绿色荧光蛋白(GFP)基因mRNA的4个靶点平行设计了4条反义寡核苷酸和4条10-23DNA酶,对照组反义寡核苷酸将最佳靶点——靶点2的反义寡核苷酸突变2个碱基,对照组10-23DNA酶将靶点2的10-23DNA酶结合臂中央突变2个碱基.体外4条10-23DNA酶切割mRNA的结果和相应的4条反义寡核苷酸依赖的RNaseH降解结果完全相似,细胞内4条10-23DNA酶对绿色荧光蛋白的表达抑制作用与相应的4条反义寡核苷酸相似,表明10-23DNA酶显示的最佳作用靶点同样是最佳作用效果的反义寡核苷酸结合靶.10-23DNA酶可以作为评价mRNA结构靶点有效性的新工具.
10-23 DNAzyme is one of antisense oligo-deoxynucleotides （asODNs） which actively split mRNA molecules. The split activity was used for verifying the binding validity of asODNs to different targeting sites on mRNA. For 4 sites on the mRNA of green fluorescence protein （GFP）, four asODNs and four 10-23 DNAzymes were designed in parallel. The control asODN had 2 mutation bases from asODN of the best site 2, and control 10-23 DNAzyme contained 2 mutation bases in the binding arms of the 10-23 DNAzyme to site 2. In vitro cleavage of GFP transcript by DNAzyme and asODNs dependent RNase H found that they have the same order in efficacy. After intracellular transfection of HeLa cells with 4 kinds of DNAzymes and 4 asODN, respectively, immuno-fluorescence microscopy examination indicated similar order in knockdown effects by reducing fluorescence intensity with most significant attenuation occurring for A4 and D4.The GFP knock down effectiveness by 10-23 DNAzyme were identical to those of respective asODNs as revealed by flow cytometry. These results suggested that 10-23 DNAzymes may be a novel tool for verifying the validity of target sites on gene mRNA.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2005年第5期622-627,共6页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金(30271546)
北京市自然科学基金(5033020)资助项目~~
