Improved knockdown from artificial microRNAs in an enhanced miR-155 backbone: a designer's guide to potent multi-target RNAi.
Improved knockdown from artificial microRNAs in an enhanced miR-155 backbone: a designer's guide to potent multi-target RNAi.
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DOI：
10.1093/nar/gkv1246
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发表时间：
2016-03-18
影响因子：
14.9
通讯作者：
Washbourne P
中科院分区：
文献类型：
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作者：
Fowler DK;Williams C;Gerritsen AT;Washbourne P
嵌入天然microRNA(MiRNA)骨架的人工microRNA(AmiRNA)序列已被证明是进行RNA干扰(RNAi)的有用工具。与短发夹状RNA(ShRNA)等其他基于RNAi的方法相比，amiRNAs减少了非靶标和毒性效应。然而，与其shRNA对应物相比，amiRNA在击倒方面往往效率较低。我们在合成抑制性BIC/miR-155 RNA(SIBR)支架上筛选了一个大型的经验性设计的amiRNA集，并显示了与有效的amiRNA相关的共同结构和序列特异性特征。然后，我们将外源基序引入到基茎区域，从而增加了amiRNA的生物发生和击倒效力。我们将这种改进的主干称为增强型SIBR(ESIBR)支架。使用连锁的amiRNAs进行多基因敲除，我们证明了针对不同基因的miRNAs的串联本身就足以提高敲除效率。此外，我们还表明，当amiRNAs链接时，eSIBR的性能优于野生型SIBR(WtSIBR)。最后，我们在培养的神经元中使用慢病毒表达系统，我们再次发现eSIBR amiRNAs对内源基因的多靶点敲除更有效。ESIBR将是RNAi方法的一个有价值的工具，特别是对于需要击倒多个靶点的研究。
Artificial microRNA (amiRNA) sequences embedded in natural microRNA (miRNA) backbones have proven to be useful tools for RNA interference (RNAi). amiRNAs have reduced off-target and toxic effects compared to other RNAi-based methods such as short-hairpin RNAs (shRNA). amiRNAs are often less effective for knockdown, however, compared to their shRNA counterparts. We screened a large empirically-designed amiRNA set in the synthetic inhibitory BIC/miR-155 RNA (SIBR) scaffold and show common structural and sequence-specific features associated with effective amiRNAs. We then introduced exogenous motifs into the basal stem region which increase amiRNA biogenesis and knockdown potency. We call this modified backbone the enhanced SIBR (eSIBR) scaffold. Using chained amiRNAs for multi-gene knockdown, we show that concatenation of miRNAs targeting different genes is itself sufficient for increased knockdown efficacy. Further, we show that eSIBR outperforms wild-type SIBR (wtSIBR) when amiRNAs are chained. Finally, we use a lentiviral expression system in cultured neurons, where we again find that eSIBR amiRNAs are more potent for multi-target knockdown of endogenous genes. eSIBR will be a valuable tool for RNAi approaches, especially for studies where knockdown of multiple targets is desired.