Establishment of human and mammalian RNAi for effective and systematic functional genomics.

本研究利用4个外源和2个内源基因的62个靶点和3个哺乳动物细胞，广泛分析了短干扰RNA(SiRNA)序列与RNA干扰(RNAi)效应的关系。我们提出了控制siRNA序列偏好的规则，并根据这些规则可以很容易地设计出系统的哺乳动物功能基因组学所必需的高效siRNA。这些规则表明，同时满足以下所有四个序列条件的siRNA能够在哺乳动物细胞中诱导高效的基因沉默：(I)反义链5‘端的A/U；(Ii)正义链5’端的G/C；(Iii)反义链5‘端三分之一的至少’5个A/U残基；以及(Iv)没有任何长度超过9个核苷酸的GC片段。在哺乳动物细胞中，与前三种条件特征相反的siRNA几乎不会引起基因沉默。除了这些siRNA序列分析外，我们还构建了一个新的用于siRNA筛选的网站。SiDirect是一个基于网络的在线软件系统，用于计算对哺乳动物RNA干扰(RNAi)具有最大靶标特异性的高效siRNA序列。使用上述新的指南选择高效的siRNA序列。我们的软件取消了非靶标基因沉默，以列举广泛使用的BLAST搜索可能忽略的潜在杂交候选基因。该网站接受任意序列作为输入，并快速返回siRNA，并在哺乳动物RNAi中提供了广泛的应用，包括系统功能基因组学和治疗性基因沉默。

In the present study, the relationship between short interfering RNA (siRNA) sequence and RNA interference (RNAi) effect was extensively analyzed using 62 targets of four exogenous and two endogenous genes and three mammalian cells. We present the rules that may govern siRNA sequence preference and in accordance with which highly effective siRNAs essential for systematic mammalian functional genomics can be readily designed. These rules indicate that siRNAs which simultaneously satisfy all four of the following sequence conditions are capable of inducing highly effective gene silencing in mammalian cells: (i) A/U at the 5' end of the antisense strand; (ii)G/C at the 5' end of the sense strand; (iii) at least 'five A/U residues in the 5' terminal one-third of the antisense strand; and (iv) the absence of any GC stretch of more than 9 nt in length. siRNAs opposite in features with respect to the first three conditions give rise to little or no gene silencing in mammalian cells. Essentially the same rules for siRNA sequence preference were found applicable to DNA-based RNAi in mammalian cells and in ovo RNAi using chick embryos.In addition to these siRNA sequence analysis, we also constructed a new web site for siRNA screening. siDirect is a web-based online software system for computing highly effective siRNA sequences with maximum target-specificity for mammalian RNA interference (RNAi). Highly effective siRNA sequences are selected using novel guidelines described above. Our software voids off-target gene silencing to enumerate potential cross-hybridization candidates that the widely used BLAST search may overlook. The website accepts an arbitrary sequence as an input and quickly returns siRNA andiates a wide scope of applications in mammalian RNAi, including systematic functional genomics and therapeutic gene silencing.