A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites.
A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites.
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对所有单核苷酸不匹配的靶位点，对活性siRNA的沉默效应进行系统分析。
DOI：
10.1093/nar/gki312
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发表时间：
2005
影响因子：
14.9
通讯作者：
Liang, ZC
中科院分区：
文献类型：
--
作者：
Du, Q;Thonberg, H;Wang, J;Wahlestedt, C;Liang, ZC
小干扰RNA（siRNA）介导的基因沉默的特异性是RNA干扰（RNAi）应用的一个关键考量因素。虽然siRNA潜在的脱靶效应的发现令人担忧，但尚未进行系统分析来探究RNAi的特异性。在此，我们介绍一项研究，其中对一种功能经过验证的siRNA（siCD46）在所有可能的57个排列的靶位点上的沉默特异性进行了检测，每个靶位点都携带一个单核苷酸突变，当与siRNA反义链配对时会产生错配。我们发现，不仅错配碱基对的位置，而且形成错配的核苷酸的种类都会影响沉默。令人惊讶的是，除了G:U摆动碱基对外，腺嘌呤（A）和胞嘧啶（C）之间形成的错配也具有良好的耐受性，含有此类错配的靶位点被siCD46沉默的效率几乎与完全匹配的靶位点相同。Northern杂交表明，含有突变靶位点的融合基因的沉默涉及靶mRNA的降解。这项研究提供了直接证据，表明siRNA的靶标识别比之前所认为的更具简并性。这一发现有助于理解RNAi的特异性，并可能有助于RNA二级结构的计算预测。
The specificity of small interfering RNA (siRNA)-mediated gene silencing is a critical consideration for the application of RNA interference (RNAi). While the discovery of potential off-target effects by siRNAs is of concern, no systematic analysis has been conducted to explore the specificity of RNAi. Here, we present a study where a functionally validated siRNA (siCD46) was examined for silencing specificity on all possible 57 permutated target sites, each carrying a single-nucleotide mutation that would generate a mismatch when paired with siRNA antisense strand. We found that it was not only the position of the mismatched base pair, but also the identity of the nucleotides forming the mismatch that influenced silencing. Surprisingly, mismatches formed between adenine (A) and cytosine (C), in addition to the G:U wobble base pair, were well tolerated and target sites containing such mismatches were silenced almost as efficiently as its fully matched counterpart by siCD46. Northern blots showed that the silencing of fusion genes harboring the mutated target sites involved target mRNA degradation. This study provides direct evidence that the target recognition of siRNA is far more degenerative than previously considered. This finding is instrumental in the understanding of RNAi specificity and may aid the computational prediction of RNA secondary structure.
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