siRNA carrying an (E)-vinylphosphonate moiety at the 5΄ end of the guide strand augments gene silencing by enhanced binding to human Argonaute-2.
siRNA carrying an (E)-vinylphosphonate moiety at the 5΄ end of the guide strand augments gene silencing by enhanced binding to human Argonaute-2.
复制标题
DOI：
10.1093/nar/gkw1298
复制
发表时间：
2017-05-05
影响因子：
14.9
通讯作者：
Joshua-Tor L
中科院分区：
文献类型：
--
作者：
Elkayam E;Parmar R;Brown CR;Willoughby JL;Theile CS;Manoharan M;Joshua-Tor L
通过RNA干扰（RNAi）在体内进行有效的基因沉默需要Argonaute蛋白识别并结合siRNA引导链的5 ′-磷酸。然而，对于外源性siRNA，它受到代谢酶快速去除引导链的5 ′-磷酸的限制。在这里，我们已经确定了与代谢稳定的5-N-（E）-乙烯基膦酸酯（5-N-E-VP）指导RNA复合的人Argonaute-2的晶体结构，分辨率为2.5-nm。该结构证明了人Argonaute-2的Mid结构域中的5 ′结合位点如何能够调节5 ′-核苷酸结合口袋中的关键残基以补偿由修饰的核苷酸引入的变化。该观察结果还解释了5-E-VP修饰的siRNA与人Argonaute-2体外结合亲和力的改善，以及在小鼠中相对于未修饰的siRNA，三价N-乙酰半乳糖胺（GalNAc）缀合的siRNA的情况下沉默的增强。
Efficient gene silencing by RNA interference (RNAi)in vivorequires the recognition and binding of the 5΄- phosphate of the guide strand of an siRNA by the Argonaute protein. However, for exogenous siRNAs it is limited by the rapid removal of the 5΄- phosphate of the guide strand by metabolic enzymes. Here, we have determined the crystal structure of human Argonaute-2 in complex with the metabolically stable 5΄-(E)-vinylphosphonate (5΄-E-VP) guide RNA at 2.5-Å resolution. The structure demonstrates how the 5΄ binding site in the Mid domain of human Argonaute-2 is able to adjust the key residues in the 5΄-nucleotide binding pocket to compensate for the change introduced by the modified nucleotide. This observation also explains improved binding affinity of the 5΄-E-VP -modified siRNA to human Argonaute-2in-vitro, as well as the enhanced silencing in the context of the trivalentN-acetylgalactosamine (GalNAc)-conjugated siRNA in mice relative to the un-modified siRNA.