siDirect 2.0: updated software for designing functional siRNA with reduced seed-dependent off-target effect.
siDirect 2.0: updated software for designing functional siRNA with reduced seed-dependent off-target effect.
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DOI：
10.1186/1471-2105-10-392
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发表时间：
2009-11-30
影响因子：
3
通讯作者：
Ui-Tei K
中科院分区：
文献类型：
--
作者：
Naito Y;Yoshimura J;Morishita S;Ui-Tei K
RNA干扰（RNAi）是由21个核苷酸（nt）长度的小干扰RNA （sirna）介导的，是研究基因功能和治疗应用的有力工具。RNAi需要siRNA引导链和目标mRNA之间的完美互补，被认为是非常特异性的。然而，最近越来越多的证据表明，siRNA可以下调转录本与7-nt siRNA种子区互补的非预期基因。这种脱靶基因沉默可能经常提供从敲低实验中获得的不一致的结果，从而导致误解。因此，基于机制特征设计具有最小脱靶效应的功能性sirna的有效算法被认为是有价值的。我们提出了siDirect 2.0，这是我们基于web的软件siDirect的更新，它为哺乳动物RNAi提供了功能性和脱靶最小化的siRNA设计。我们的前一个版本的软件通过考虑siRNA序列和RNAi活性之间的关系来设计功能性siRNA，并通过快速灵敏的同源搜索算法提供潜在脱靶候选基因的计数。在新版本中，siRNA设计算法进行了广泛的更新，以消除脱靶效应，这反映了我们最近的发现，即siRNA诱导脱靶效应的能力与种子-靶双链的热力学稳定性或熔融温度（Tm）高度相关，种子-靶双链是在siRNA引导链5'端2-8位的核苷酸与其靶mRNA之间形成的。选择具有较低种子-靶标双工稳定性（基准Tm < 21.5°C）的sirna，然后消除几乎完全匹配的不相关转录本，可以最大限度地减少脱靶效应。siDirect 2.0通过更新的算法提供功能性的靶向siRNA设计，大大减少了脱靶沉默。当候选功能siRNA能够形成Tm值低于21.5°C的种子靶双链，并且它们的19-nt区域跨越两条链的2-20位与任何其他非靶向转录物至少有两个错配时，siDirect 2.0可以设计至少一个符合条件的siRNA，用于RefSeq中b> 94%的人类mRNA序列。siDirect 2.0可从http://siDirect2.RNAi.jp/获得。
RNA interference (RNAi), mediated by 21-nucleotide (nt)-length small interfering RNAs (siRNAs), is a powerful tool not only for studying gene function but also for therapeutic applications. RNAi, requiring perfect complementarity between the siRNA guide strand and the target mRNA, was believed to be extremely specific. However, a recent growing body of evidence has suggested that siRNA could down-regulate unintended genes whose transcripts possess complementarity to the 7-nt siRNA seed region. This off-target gene silencing may often provide incongruous results obtained from knockdown experiments, leading to misinterpretation. Thus, an efficient algorithm for designing functional siRNAs with minimal off-target effect based on the mechanistic features is considered of value. We present siDirect 2.0, an update of our web-based software siDirect, which provides functional and off-target minimized siRNA design for mammalian RNAi. The previous version of our software designed functional siRNAs by considering the relationship between siRNA sequence and RNAi activity, and provided them along with the enumeration of potential off-target gene candidates by using a fast and sensitive homology search algorithm. In the new version, the siRNA design algorithm is extensively updated to eliminate off-target effects by reflecting our recent finding that the capability of siRNA to induce off-target effect is highly correlated to the thermodynamic stability, or the melting temperature (Tm), of the seed-target duplex, which is formed between the nucleotides positioned at 2-8 from the 5' end of the siRNA guide strand and its target mRNA. Selection of siRNAs with lower seed-target duplex stabilities (benchmark Tm < 21.5°C) followed by the elimination of unrelated transcripts with nearly perfect match should minimize the off-target effects. siDirect 2.0 provides functional, target-specific siRNA design with the updated algorithm which significantly reduces off-target silencing. When the candidate functional siRNAs could form seed-target duplexes with Tm values below 21.5°C, and their 19-nt regions spanning positions 2-20 of both strands have at least two mismatches to any other non-targeted transcripts, siDirect 2.0 can design at least one qualified siRNA for >94% of human mRNA sequences in RefSeq. siDirect 2.0 is available at http://siDirect2.RNAi.jp/.
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