Tanmay Chatterjee, Shankar Mandal, Sujay Ray, Alexander Johnson‐Buck, Nils G. Walter
Silencing by the miRNA-guided RNA induced silencing complex (miRISC) is dependent on Ago2-chaperoned base pairing between the miRNA 5′ seed (5′S) and a complementary sequence in the 3′ untranslated region of an mRNA. Prevailing mechanistic understanding posits that initial 5′S pairing can further allow functional base pair expansion into the 3′ non-seed (3′NS), while functionally distinct non-canonical pairing was reported between only the 3′NS and the mRNA coding sequence. We developed single-molecule kinetics through equilibrium Poisson sampling (SiMKEPS) to measure highly precise binding and dissociation rate constants of varying-length target sequences to 5′S and 3′NS in a paradigmatic miRISC isolated from human cells, revealing distinct stable states of miRISC with mutually exclusive 5′S and 3′NS pairing. Our data suggests conformational rearrangements of the Ago2-bound miRNA that regulate alternative 5′S- and 3′NS-driven target recognition. The resulting model reconciles previously disparate observations and deepens our acumen for successfully marshaling RNA silencing therapies. MicroRNAs regulate gene expression through selective pairing with target mRNAs. Here, the authors reveal distinct, mutually exclusive binding modes of miRISC at the 5′ seed and 3′ non-seed regions, advancing understanding of RNA silencing mechanisms.