Center of Excellence in Infectious Disease Research, Department of Biomedical Sciences, Paul L Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, Texas, United States of America.
PLoS One. 2011;6(12):e28580. doi: 10.1371/journal.pone.0028580. Epub 2011 Dec 9.
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.
siRNA（小干扰 RNA）和 shRNA（短发夹 RNA）是生物医学研究中强大且常用的工具。目前，siRNA 通常设计为两条 21 个核苷酸的 RNA 链，其中包括 19 个完全互补的核苷酸和 2 个核苷酸的突出端。然而，由于 si/shRNA 利用内源性 miRNA 机制进行基因沉默，而 miRNA 通常长 22 个核苷酸并包含多个内部错配，因此我们测试了通过设计 si/shRNA 来模拟 miRNA 结构是否可以提高其功能。我们系统地研究了在不同位置引入过客链中单或多个错配对 siRNA 功能的影响。在某些位置的错配可以显著提高 siRNA 的功能，并且在某些情况下降低了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，具有或不具有过客链中错配的 si 和 miRNA 结构的寡核苷酸（siRNA）都可以抑制所有含有 Ago 的细胞中的靶标，这表明 Ago 蛋白不会区分用于沉默活性的 si/miRNA 结构。
