The guide RNA sequence dictates the slicing kinetics and conformational dynamics of the Argonaute silencing complex. Wang et al.

Published: 7 July 2024| Version 2 | DOI: 10.17632/j82nkmfppj.2


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.



Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Howard Hughes Medical Institute


Biochemistry, Biophysics, microRNA, RNA Interference