Construction of siRNA library for human functional genomics and hunting of RNAi-related genes.

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RNA interference (RNAi) has been shown quite useful in the clarification of gene function in various organisms. Synthetic 21bp long double-stranded RNAs (dsRNAs) each with two 2nt long 3'overhangs have been found to serve as short interfering RNAs (siRNAs) for mammalian RNAi. The mechanisms of 21bp-long siRNA dependent RNAi in mammalian cells have been studied extensively, and we have established the rules for designing sequences of highly effective siRNAs. Our guidelines indicate 21bp long siRNAs simultaneously satisfying all four of the following sequence conditions to be capable of inducing highly effective gene-silencing in mammalian cells : A/U at the 5'end of the guide strand (GS) ; G/C at the 5'end of the passenger strand (PS) ; at least four A/U residues in the 5'terminal third of GS and the absence of any GC stretch of more than 9nt in length. RNAi is also known to be induced by the transfection of DNA encoding short hairpin RNA (shRNA). Large scale screening of loss-of-functi … More on mutants is possible if suitable shRNA-encoding DNA libraries are available. For construction of an shRNA-encoding DNA library, RNA-polymerase-III-promoter driven vectors have been widely used. But, this pol-III-driven system imposes various restrictions on shRNA sequences so that a considerable fraction of siRNA sequences becomes unavailable. To surmount this difficulty, we developed a new vector system which is driven by RNA polymerase II promoter. It is now possible to design any effective shRNA-encoding DNA without any sequence restrictions. I addition, we found that not only 21bp siRNA but also 22bp dsRNA is the final Dicer digestion product and showed some fraction of 22bp dsRNA to serve as an effective siRNA. Sequence preference rules for highly effective 22bp siRNA were very similar, if not identical, to those for 21bp siRNAs. We also found siRNA dimer and trimer to be capable of efficiently inducing RNAi when these oligomers possess two 2nt-long 3'overhangs and contain an active monomer unit in frame with respect to Dicer digestion. Thus, double- or triple-knockdown is possible in some suitable cell lines. Finally, we carried out gene screening experiments using our siRNA libraries and identified many candidates for human or mouse transcription-factor genes, apoptosis-related genes, and RNAi-related genes. Less