Competition for RISC binding predicts in vitro potency of siRNA.
Competition for RISC binding predicts in vitro potency of siRNA.
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DOI：
10.1093/nar/gkl589
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发表时间：
2006
影响因子：
14.9
通讯作者：
Dean NM
中科院分区：
文献类型：
--
作者：
Koller E;Propp S;Murray H;Lima W;Bhat B;Prakash TP;Allerson CR;Swayze EE;Marcusson EG;Dean NM
短干扰RNA（siRNA）通过RNA诱导的沉默复合物（RISC）引导靶RNA的降解。siRNA在动物中的使用受到限制，部分原因是siRNA在组织中的半衰期短。化学修饰的siRNA是维持mRNA降解活性所必需的，但对核酸酶更稳定。在这项研究中，我们利用交替的2′-O-甲基和2′-脱氧-2 ′-氟（OMe/F）化学修饰的siRNA靶向PTEN和Eg 5。OMe/F修饰的siRNA以与未修饰的siRNA相同或更高的效力和功效一致地降低mRNA和蛋白质水平。我们发现，修饰的siRNA使用RISC机制，并导致在与未修饰的siRNA相同的位置切割靶mRNA。我们进一步证明了siRNA可以相互竞争，其中高效siRNA可以与低效siRNA竞争，从而限制了具有较低效力的siRNA介导mRNA降解的能力。相反，具有低效力的siRNA不能与高效siRNA竞争。我们建立了siRNA效力和与其他siRNA竞争的能力之间的相关性。因此，作为mRNA破坏的更有效抑制剂的siRNA具有胜过较低效siRNA的潜力，这表明细胞组分（可能是RISC）的量限制了siRNA活性。
Short interfering RNAs (siRNA) guide degradation of target RNA by the RNA-induced silencing complex (RISC). The use of siRNA in animals is limited partially due to the short half-life of siRNAs in tissues. Chemically modified siRNAs are necessary that maintain mRNA degradation activity, but are more stable to nucleases. In this study, we utilized alternating 2′-O-methyl and 2′-deoxy-2′-fluoro (OMe/F) chemically modified siRNA targeting PTEN and Eg5. OMe/F-modified siRNA consistently reduced mRNA and protein levels with equal or greater potency and efficacy than unmodified siRNA. We showed that modified siRNAs use the RISC mechanism and lead to cleavage of target mRNA at the same position as unmodified siRNA. We further demonstrated that siRNAs can compete with each other, where highly potent siRNAs can compete with less potent siRNAs, thus limiting the ability of siRNAs with lower potency to mediate mRNA degradation. In contrast, a siRNA with low potency cannot compete with a highly efficient siRNA. We established a correlation between siRNA potency and ability to compete with other siRNAs. Thus, siRNAs that are more potent inhibitors for mRNA destruction have the potential to out-compete less potent siRNAs indicating that the amount of a cellular component, perhaps RISC, limits siRNA activity.
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