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

建立人类和哺乳动物 RNAi，以实现有效和系统的功能基因组学。

基本信息

批准号：
13358012
负责人：
SAIGO Kaoru
金额：
$ 22.21万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

项目摘要

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.

在本研究中，使用四个外源基因和两个内源基因的62个靶标以及三个哺乳动物细胞广泛分析了短干扰RNA（siRNA）序列和RNA干扰（RNAi）效应之间的关系。我们提出了可能控制 siRNA 序列偏好的规则，并根据这些规则可以轻松设计系统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) 有义链 5' 端的 G/C； (iii)反义链的5'端三分之一处至少有“五个A/U残基； (iv) 不存在任何长度超过 9 nt 的 GC 延伸。与前三种条件特征相反的 siRNA 在哺乳动物细胞中几乎不产生或不产生基因沉默。基本上相同的 siRNA 序列偏好规则适用于哺乳动物细胞中基于 DNA 的 RNAi 和使用鸡胚的卵内 RNAi。除了这些 siRNA 序列分析之外，我们还构建了一个用于 siRNA 筛选的新网站。 siDirect 是一个基于网络的在线软件系统，用于计算高效的 siRNA 序列，对哺乳动物 RNA 干扰 (RNAi) 具有最大的靶标特异性。使用上述新颖的指导原则来选择高效的 siRNA 序列。我们的软件避免了脱靶基因沉默，以枚举广泛使用的 BLAST 搜索可能忽略的潜在交叉杂交候选者。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 中具有广泛的应用，包括系统功能基因组学和治疗性基因沉默。