Ribozyme-enhanced single-stranded Ago2-processed interfering RNA triggers efficient gene silencing with fewer off-target effects.
Ribozyme-enhanced single-stranded Ago2-processed interfering RNA triggers efficient gene silencing with fewer off-target effects.
复制标题
核酶增强的单链 Ago2 处理的干扰 RNA 可触发有效的基因沉默，同时减少脱靶效应
DOI：
10.1038/ncomms9430
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发表时间：
2015-10-12
影响因子：
16.6
通讯作者：
Wu L
中科院分区：
文献类型：
--
作者：
Shang R;Zhang F;Xu B;Xi H;Zhang X;Wang W;Wu L
短发夹RNA（Short-hairpin RNA，shRNAs）被广泛用于产生小干扰RNA（small-interfering RNA，siRNAs），用于基因沉默。在这里，我们设计了一种替代的siRNA前体，命名为单链，Argonaute 2（Ago 2）加工的干扰RNA（saiRNA），含有一个16-18 bp的茎和一个环互补的目标转录。将来自丁型肝炎病毒的自切割核酶引入到转录的saiRNA的3 '末端，通过产生促进saiRNA与Ago 2有效结合的短3'突出端，显著提高了其沉默活性。相同的核酶也增强Dicer依赖性shRNA的活性。与经典的shRNA不同，在加工过程中Ago 2对saiRNA的链特异性切割消除了过客链并防止siRNA与非溶核Ago蛋白的缔合。因此，减少了脱靶效应。此外，saiRNA表现出与内源性miRNA的生物合成的竞争较小。因此，核酶增强的saiRNA为RNA干扰应用提供了可靠的工具。 短发夹RNA被广泛用于产生用于基因沉默的小干扰RNA（siRNA）。在这里，作者表明，在自切割核酶存在下的替代siRNA前体具有增强的沉默活性和减少的脱靶效应，提供了一种潜在的RNAi工具。
Short-hairpin RNAs (shRNAs) are widely used to produce small-interfering RNAs (siRNAs) for gene silencing. Here we design an alternative siRNA precursor, named single-stranded, Argonaute 2 (Ago2)-processed interfering RNA (saiRNA), containing a 16–18 bp stem and a loop complementary to the target transcript. The introduction of a self-cleaving ribozyme derived from hepatitis delta virus to the 3′ end of the transcribed saiRNA dramatically improves its silencing activity by generating a short 3′ overhang that facilitates the efficient binding of saiRNA to Ago2. The same ribozyme also enhances the activity of Dicer-dependent shRNAs. Unlike a classical shRNA, the strand-specific cleavage of saiRNA by Ago2 during processing eliminates the passenger strand and prevents the association of siRNA with non-nucleolytic Ago proteins. As a result, off-target effects are reduced. In addition, saiRNA exhibits less competition with the biogenesis of endogenous miRNAs. Therefore, ribozyme-enhanced saiRNA provides a reliable tool for RNA interference applications. Short hairpin RNAs are widely used to produce small interfering RNAs (siRNAs) for gene silencing. Here, the authors show that an alternative siRNA precursor in the presence of a self-cleaving ribozyme has enhanced silencing activity and reduced off-target effects, providing a potential RNAi tool.