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

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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.