Improved siRNA/shRNA functionality by mismatched duplex.
Improved siRNA/shRNA functionality by mismatched duplex.
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DOI：
10.1371/journal.pone.0028580
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Manjunath N
中科院分区：
文献类型：
--
作者：
Wu H;Ma H;Ye C;Ramirez D;Chen S;Montoya J;Shankar P;Wang XA;Manjunath N
siRNA（small interfering RNA）和shRNA（small hairpin RNA）是生物医学研究中常用的有力工具。目前，siRNA通常被设计为两条21 nt的RNA链，其包括19 nt完全互补的部分和2 nt突出端。然而，由于si/shRNAs使用内源性miRNA机制进行基因沉默，并且miRNA的长度通常为22 nt并且含有多个内部错配，因此我们测试了是否可以通过设计si/shRNAs以模拟miRNA结构来增加功能性。我们系统地研究了在乘客链的不同位置处引入的单个或多个错配对siRNA功能的影响。某些位置的错配可以显著增加siRNA的功能，并且在某些情况下还可以降低不需要的过客链功能。同样的策略也可以用于设计shRNA。最后，我们表明si和miRNA结构的寡核苷酸（在过客链中具有或不具有错配的siRNA）都可以抑制所有含有Ago的细胞中的靶标，这表明Ago蛋白不区分基于si/miRNA的结构的沉默活性。
siRNA (small interfering RNA) and shRNA (small hairpin RNA) are powerful and commonly used tools in biomedical research. Currently, siRNAs are generally designed as two 21 nt strands of RNA that include a 19 nt completely complementary part and a 2 nt overhang. However, since the si/shRNAs use the endogenous miRNA machinery for gene silencing and the miRNAs are generally 22 nt in length and contain multiple internal mismatches, we tested if the functionality can be increased by designing the si/shRNAs to mimic a miRNA structure. We systematically investigated the effect of single or multiple mismatches introduced in the passenger strand at different positions on siRNA functionality. Mismatches at certain positions could significantly increase the functionality of siRNAs and also, in some cases decreased the unwanted passenger strand functionality. The same strategy could also be used to design shRNAs. Finally, we showed that both si and miRNA structured oligos (siRNA with or without mismatches in the passenger strand) can repress targets in all individual Ago containing cells, suggesting that the Ago proteins do not differentiate between si/miRNA-based structure for silencing activity.