Functional polarity is introduced by Dicer processing of short substrate RNAs.
Functional polarity is introduced by Dicer processing of short substrate RNAs.
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DOI：
10.1093/nar/gki732
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发表时间：
2005
影响因子：
14.9
通讯作者：
Behlke MA
中科院分区：
文献类型：
--
作者：
Rose SD;Kim DH;Amarzguioui M;Heidel JD;Collingwood MA;Davis ME;Rossi JJ;Behlke MA
作为Dicer底物的合成RNA双链体是RNA干扰（RNAi）的有效触发物。平端27聚体双链体的效力比传统的21聚体双链体高100倍。并非所有27聚体双链体都显示出增加的效力。使用电喷雾电离质谱法对体外切割反应的产物的评价揭示了多种产物可以通过Dicer切割产生。使用具有单个2-碱基3′-突出端的不对称双链体限制了切割导致的异质性。在平端双链末端包含DNA残基也限制了异质性。不对称的2-碱基3′-突出端与平端上的3′-DNA残基的组合产生双链体形式，其指导切割以可预测地产生单个初级切割产物。因此，可以设计27聚体双链体，其通过Dicer加工以产生特定的所需21聚体种类。使用这种策略，可以设计两种不同的27聚体，其在切割后产生相同的21聚体，一种是3’-突出端位于反义（AS）链上，切割向‘右’（‘R’）进行，另一种是3’-突出端位于有义（S）链上，切割向‘左’（‘L’）进行。有趣的是，不对称27聚体的“R”版本通常比“L”版本27聚体在降低靶基因水平方面更有效。链靶向实验显示了两种不同的27聚体形式之间的不对称链利用，其中“R”形式有利于S链，而“L”形式有利于AS链沉默。因此，Dicer加工赋予RNAi途径内的功能极性。
Synthetic RNA duplexes that are substrates for Dicer are potent triggers of RNA interference (RNAi). Blunt 27mer duplexes can be up to 100-fold more potent than traditional 21mer duplexes. Not all 27mer duplexes show increased potency. Evaluation of the products of in vitro dicing reactions using electrospray ionization mass spectrometry reveals that a variety of products can be produced by Dicer cleavage. Use of asymmetric duplexes having a single 2-base 3′-overhang restricts the heterogeneity that results from dicing. Inclusion of DNA residues at the ends of blunt duplexes also limits heterogeneity. Combination of asymmetric 2-base 3′-overhang with 3′-DNA residues on the blunt end result in a duplex form which directs dicing to predictably yield a single primary cleavage product. It is therefore possible to design a 27mer duplex which is processed by Dicer to yield a specific, desired 21mer species. Using this strategy, two different 27mers can be designed that result in the same 21mer after dicing, one where the 3′-overhang resides on the antisense (AS) strand and dicing proceeds to the ‘right’ (‘R’) and one where the 3′-overhang resides on the sense (S) strand and dicing proceeds to the ‘left’ (‘L’). Interestingly, the ‘R’ version of the asymmetric 27mer is generally more potent in reducing target gene levels than the ‘L’ version 27mer. Strand targeting experiments show asymmetric strand utilization between the two different 27mer forms, with the ‘R’ form favoring S strand and the ‘L’ form favoring AS strand silencing. Thus, Dicer processing confers functional polarity within the RNAi pathway.
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影响因子：
11.4
作者：
Elbashir, SM;Martinez, J;Tuschl, T
通讯作者：
Tuschl, T
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作者：
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作者：
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作者：
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