Whole-genome thermodynamic analysis reduces siRNA off-target effects.
Whole-genome thermodynamic analysis reduces siRNA off-target effects.
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DOI：
10.1371/journal.pone.0058326
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Chou HH
中科院分区：
文献类型：
--
作者：
Chen X;Liu P;Chou HH
小干扰RNA(SiRNAs)是敲除靶基因的重要工具，已广泛应用于生物和生物医学研究。为了设计siRNAs，必须考虑两个重要方面：击倒靶基因的效力和对任何非靶基因的脱靶效应。虽然许多研究已经产生了有用的工具来设计有效的siRNA，但脱靶预防大多被委托给序列水平的比对工具，如BLAST。我们假设全基因组热力学分析可以更精确地识别潜在的脱靶效应，并帮助我们避免可能具有强烈脱靶效应的siRNAs。为了验证这一假设，设计了两组针对三个人类基因IDH1、ITPR2和TRIM28的siRNA。它们是从两个流行的siRNA设计工具siDirect和siDesign的输出中挑选出来的。这两种siRNA设计工具都包含了序列水平的筛选，以避免脱靶，因此它们的输出被认为是最佳的。然而，我们测试的其中一组基因有偏离目标的基因，这是由全基因组热力学分析工具Picky预测的。Picky可以识别可能在用户指定的熔化温度范围内与siRNA杂交的非靶标基因。我们的实验证实，Picky预测的一些非靶标基因确实可以被siRNAs抑制。使用商业上可用的siRNA进行了类似的实验，也发现一些非靶标基因也被抑制，正如Picky预测的那样。综上所述，我们证明了全基因组热力学分析可以识别序列水平筛选中遗漏的非靶标基因。因为根据热力学，挑剔的预测是确定性的，如果候选的siRNA没有挑剔的预测脱靶，就不太可能引起脱靶效应。因此，我们建议在siRNA设计中将挑剔作为额外的筛选步骤。
Small interfering RNAs (siRNAs) are important tools for knocking down targeted genes, and have been widely applied to biological and biomedical research. To design siRNAs, two important aspects must be considered: the potency in knocking down target genes and the off-target effect on any nontarget genes. Although many studies have produced useful tools to design potent siRNAs, off-target prevention has mostly been delegated to sequence-level alignment tools such as BLAST. We hypothesize that whole-genome thermodynamic analysis can identify potential off-targets with higher precision and help us avoid siRNAs that may have strong off-target effects. To validate this hypothesis, two siRNA sets were designed to target three human genes IDH1, ITPR2 and TRIM28. They were selected from the output of two popular siRNA design tools, siDirect and siDesign. Both siRNA design tools have incorporated sequence-level screening to avoid off-targets, thus their output is believed to be optimal. However, one of the sets we tested has off-target genes predicted by Picky, a whole-genome thermodynamic analysis tool. Picky can identify off-target genes that may hybridize to a siRNA within a user-specified melting temperature range. Our experiments validated that some off-target genes predicted by Picky can indeed be inhibited by siRNAs. Similar experiments were performed using commercially available siRNAs and a few off-target genes were also found to be inhibited as predicted by Picky. In summary, we demonstrate that whole-genome thermodynamic analysis can identify off-target genes that are missed in sequence-level screening. Because Picky prediction is deterministic according to thermodynamics, if a siRNA candidate has no Picky predicted off-targets, it is unlikely to cause off-target effects. Therefore, we recommend including Picky as an additional screening step in siRNA design.
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作者：
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通讯作者：
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发表时间：
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影响因子：
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作者：
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