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. HIGHLIGHTS • 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 ### Competing Interest Statement D.P.B. has equity in Alnylam Pharmaceuticals, where he is a co-founder and advisor. P.Y.W. declares no competing interests.