Increased siRNA duplex stability correlates with reduced off-target and elevated on-target effects
Increased siRNA duplex stability correlates with reduced off-target and elevated on-target effects
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DOI：
10.1261/rna.2348111
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发表时间：
2011-04-01
期刊：
影响因子：
4.5
通讯作者：
Meister, Gunter
中科院分区：
文献类型：
--
作者：
Petri, Sebastian;Dueck, Anne;Meister, Gunter
Argonaute（Ago）蛋白形成RNA诱导的沉默复合物（RISC）的核心并介导小RNA引导的基因沉默。在RNAi中，短干扰RNA（siRNA）将RISC引导至互补靶RNA，导致内切核酸酶Ago 2的切割。然而，非催化Ago蛋白也有助于RNAi，但不能切割靶RNA，并且通常产生脱靶效应。在这里，我们表明，合成的siRNA双链体与所有的Ago蛋白相互作用，但一个功能RISC快速组装只有Ago 2周围。通过稳定的siRNA双链体，我们表明，非催化Ago蛋白Ago 1，-3，和-4可以被选择性地阻止，不形成功能性RISC。此外，稳定的siRNA更有效地形成Ago 2-RISC，导致沉默活性增加。我们的数据表明新的参数设计的siRNA与选择性激活的核酸内切酶Ago 2。
Argonaute (Ago) proteins form the core of RNA-induced silencing complexes (RISCs) and mediate small RNA-guided gene silencing. In RNAi, short interfering RNAs (siRNAs) guide RISCs to complementary target RNAs, leading to cleavage by the endonuclease Ago2. Noncatalytic Ago proteins, however, contribute to RNAi as well but cannot cleave target RNA and often generate off-target effects. Here we show that synthetic siRNA duplexes interact with all Ago proteins, but a functional RISC rapidly assembles only around Ago2. By stabilizing the siRNA duplex, we show that the noncatalytic Ago proteins Ago1, -3, and -4 can be selectively blocked and do not form functional RISCs. In addition, stabilized siRNAs form an Ago2-RISC more efficiently, leading to increased silencing activity. Our data suggest novel parameters for the design of siRNAs with selective activation of the endonuclease Ago2.