It has been reported that the two major types of RNA interference triggers, the classical Dicer-generated small RNAs (siRNAs), which function with all members of the Argonaute (Ago) protein family in mammals, and the Ago2-sliced small RNAs (sli-siRNAs), which function solely through Ago2, have similar potency in target cleavage and repression. Here, we show that sli-siRNAs are generally more potent than siRNAs in silencing mismatched targets. This phenomenon is usually more apparent in targets that have mismatched nucleotides in the 3 supplementary region than in targets with mismatches in the seed region. We demonstrate that Ago2 slicer activity is a major factor contributing to the greater silencing efficiency of sli-siRNA against mismatched targets and that participation of non-slicing Agos in silencing mismatched siRNA targets may dilute the slicing ability of Ago2. The difference in length of the mature guide RNA used in sli-RISCs and si-RISCs may also contribute to the observed difference in knockdown efficiency. Our data suggest that a sli-siRNA guide strand is likely to have substantially stronger off-target effects than a guide strand with the same sequence in a classical siRNA and that Dicer and non-slicing Agos may play pivotal roles in controlling siRNA target specificity.
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