Identifying Cleaved and Noncleaved Targets of Small Interfering RNAs and MicroRNAs in Mammalian Cells by SpyCLIP.
Identifying Cleaved and Noncleaved Targets of Small Interfering RNAs and MicroRNAs in Mammalian Cells by SpyCLIP.
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通过 SpyCLIP 识别哺乳动物细胞中小干扰 RNA 和 MicroRNA 的切割和非切割靶标
DOI：
10.1016/j.omtn.2020.10.009
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发表时间：
2020-12-04
期刊：
影响因子：
--
通讯作者：
Wu L
中科院分区：
文献类型：
--
作者：
Zhang Y;Teng Y;Xiao W;Xu B;Zhao Y;Li W;Wu L
近日，美国食品药品监督管理局（FDA）批准了第一个小干扰RNA（siRNA）药物，标志着RNA干扰（RNAi）技术在治疗应用上的一个重要里程碑。然而，通过siRNA的脱靶基因沉默仍然是siRNA治疗的主要障碍之一。尽管已经广泛讨论了由已知的microRNA（miRNA）介导的基因抑制机制引起的siRNA脱靶效应，但RNAi是否可以通过效应蛋白AGO2在含有与siRNA部分互补序列的位点引起非预期的切割仍然未知。在这里，我们报告了一种策略，建立一个全面的图片siRNA切割和非切割脱靶通过执行SpyCLIP使用野生型和催化失活AGO2突变体平行。此外，我们使用相同的策略研究了由内源性miRNA介导的天然发生的切割事件。我们的研究结果表明，AGO2 SpyCLIP是一个强大的方法，以确定切割和非切割的siRNA的目标，为改善siRNA的设计规则提供了有价值的信息。FDA批准patisiran和givosiran预示着siRNA药物时代的到来。然而，脱靶效应仍然是开发siRNA治疗的挑战。在此，相应的作者和同事报告了一种有效的策略来识别siRNA切割和非切割的脱靶，为改进siRNA设计规则提供了有价值的信息。
Recently, the US Food and Drug Administration (FDA) approved the first small interfering RNA (siRNA) drug, marking a significant milestone in the therapeutic use of RNA interference (RNAi) technology. However, off-target gene silencing by siRNA remains one of the major obstacles in siRNA therapy. Although siRNA off-target effects caused by a mechanism known for microRNA (miRNA)-mediated gene repression have been extensively discussed, whether RNAi can cause unintended cleavage through the effector protein AGO2 at sites harboring partially complementary sequences to the siRNA remains unknown. Here, we report a strategy to establish a comprehensive picture of siRNA cleaved and noncleaved off-targets by performing SpyCLIP using wild-type and catalytically inactive AGO2 mutants in parallel. Additionally, we investigated naturally occurring cleavage events mediated by endogenous miRNAs using the same strategy. Our results demonstrated that AGO2 SpyCLIP is a powerful method to identify both the cleaved and noncleaved targets of siRNAs, providing valuable information for improving siRNA design rules. Approval of patisiran and givosiran by the FDA heralds the arrival age of siRNA medicines. However, off-target effect remains a challenge in developing siRNA therapy. Herein, corresponding authors and colleagues report an effective strategy to identify siRNA cleaved and noncleaved off-targets, providing valuable information for improving siRNA design rules.
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