Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate
Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate
复制标题
DOI：
10.1093/emboj/20.23.6877
复制
发表时间：
2001-12-03
期刊：
影响因子：
11.4
通讯作者：
Tuschl, T
中科院分区：
文献类型：
--
作者：
Elbashir, SM;Martinez, J;Tuschl, T
21-23核苷酸（nt） RNA的双链是RNA干扰（RNAi）和转录后基因沉默（PTGS）的序列特异性介质。研究了黑腹果蝇胚胎裂解液中合成的短干扰rna （sirna）对其长度、结构、化学组成和序列的要求，以介导有效的RNAi。具有2 nt 3'悬垂的21 nt sirna双链是序列特异性mRNA降解的最有效触发器。用2'-脱氧或2‘- o -甲基寡核苷酸取代一条或两条siRNA链可以消除RNAi，尽管在siRNA的3’端可以容忍多次2'-脱氧核苷酸取代。目标识别过程具有高度的序列特异性，但并不是siRNA的所有位置都对目标识别有同样的贡献；siRNA双工中心的错配阻止了靶RNA的切割。切割位点在靶RNA中的位置是由向导siRNA的5‘端而不是3’端定义的。这些结果为今后基因靶向实验中sirna的设计提供了合理的依据。
Duplexes of 21-23 nucleotide (nt) RNAs are the sequence-specific mediators of RNA interference (RNAi) and post-transcriptional gene silencing (PTGS). Synthetic, short interfering RNAs (siRNAs) were examined in Drosophila melanogaster embryo lysate for their requirements regarding length, structure, chemical composition and sequence in order to mediate efficient RNAi. Duplexes of 21 nt siRNAs with 2 nt 3' overhangs were the most efficient triggers of sequence-specific mRNA degradation. Substitution of one or both siRNA strands by 2'-deoxy or 2'-O-methyl oligonucleotides abolished RNAi, although multiple 2'-deoxynucleotide substitutions at the 3' end of siRNAs were tolerated. The target recognition process is highly sequence specific, but not all positions of a siRNA contribute equally to target recognition; mismatches in the centre of the siRNA duplex prevent target RNA cleavage. The position of the cleavage site in the target RNA is defined by the 5' end of the guide siRNA rather than its 3' end. These results provide a rational basis for the design of siRNAs in future gene targeting experiments.