The guide-RNA sequence dictates the slicing kinetics and conformational dynamics of the Argonaute silencing complex

The RNA-induced silencing complex (RISC), which powers RNA interference (RNAi), consists of a guide RNA and an Argonaute protein that slices target RNAs complementary to the guide. We find that, for different guide-RNA sequences, slicing rates of perfectly complementary bound targets can be surprisingly different (>250-fold range), and that faster slicing confers better knockdown in cells. Nucleotide sequence identities at guide-RNA positions 7, 10, and 17 underlie much of this variation in slicing rates. Analysis of one of these determinants implicates a structural distortion at guide nucleotides 6–7 in promoting slicing. Moreover, slicing directed by different guide sequences has an unanticipated, 600-fold range in 3′-mismatch tolerance, attributable to guides with weak (AU-rich) central pairing requiring extensive 3′ complementarity (pairing beyond position 16) to more fully populate the slicing-competent conformation. Together, our analyses identify sequence determinants of RISC activity and provide biochemical and conformational rationale for their action. [Display omitted] •Sequence of guide RNA can alter slicing rate of fully paired substrate by 250-fold•Sequences that cause more rapid slicing direct more efficient RNAi in cells•Strong central pairing imparts tolerance for mismatches to the guide 3′ region•This tolerance is attributable to more fully populating the slicing conformation Wang and Bartel identify how the sequence of the guide RNA determines the rate and base-pairing requisites of AGO2-catalyzed slicing, thereby influencing RNAi-mediated knockdown. They link these determinants to the conformational changes required to achieve the slicing-competent configuration, providing insight into the biophysical determinants of AGO2 activity and the design of synthetic siRNAs.