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Human Argonaute-2 (Ago2) is loaded by target-specific guide RNA from duplex siRNA resulting in the formation of biologically active RISC.Two independent lines of experimental evidence suggest the involvement of target RNA in Argonaute-2-loading by guide RNA.Firstly, competition of siRNA-induced RNAi by heterologous siRNA requires the competitor-specific target RNA for competition.Secondly, in an artificial sequence system with no homology to human sequences, Ago2 selectively associates with the strand of duplex siRNA which is complementary to target-RNA.Our observations are consistent with a model in which activation of RISC requires a first round of target suppression/cleavage.