• Article

• Open access

• Published: 08 December 2025

• Tanmay Chatterjee1,

• Shankar Mandal1,

• Sujay Ray1,

• Alexander Johnson-Buck1 &

• …

• Nils G. Walter ORCID: orcid.org/0000-0002-7301-12751

Nature Communications , Article number: (2025) Cite this article

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Abstract

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.

Data availability

The data supporting the findings of this study are available from the corresponding authors upon request. Source data are available through Deep Blue deposit (https://doi.org/10.7302/r2en-7y96)53.

Code availability

All MATLAB scripts used for analysis of the data are deposited through Deep Blue deposit (https://doi.org/10.7302/r2en-7y96)53.

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Acknowledgements

This work was funded by NIH grant GM131922 to N.G.W. SiMREPS analysis software for kinetic fingerprinting analysis was developed in part based on funding from a Michigan Economic Development Corporation MTRAC for Life Sciences grant to N.G.W. and A.J-B. as well as NIH grants R21 CA204560 and R33 CA229023 to N.G.W. We thank Dr. Leemor Joshua-Tor and Dr. Katarina Meze for providing us with the purified empty Ago2.

Author information

Authors and Affiliations

Single Molecule Analysis Group and Center for RNA Biomedicine, Department of Chemistry, University of Michigan, Ann Arbor, MI, USA

Tanmay Chatterjee, Shankar Mandal, Sujay Ray, Alexander Johnson-Buck & Nils G. Walter

Authors

1. Tanmay Chatterjee
2. Shankar Mandal
3. Sujay Ray
4. Alexander Johnson-Buck
5. Nils G. Walter

Contributions

T.C., S.M., and N.G.W. conceived the idea and devised the approach for kinetic fingerprinting-based accessibility detection of guide strand of miRISC. T.C., S.M., and S.R. designed the experiments, collected, and analyzed the data, and wrote the initial draft of the manuscript. A.J.B. performed the trace simulations and single-trace kinetic analyses. All authors interpreted the data and edited the manuscript.

Corresponding author

Correspondence to Nils G. Walter.

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Competing interests

The authors declare the following competing financial interests: A.J.-B. and N.G.W. are inventors on multiple patent applications related to SiMKEPS, and equity holders of aLight Sciences Inc., a startup company aiming to commercialize related technology. The remaining authors declare no competing interests.

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Nature Communications thanks Chirlmin Joo, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

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Chatterjee, T., Mandal, S., Ray, S. et al. A unifying model for microRNA-guided silencing of messenger RNAs. Nat Commun (2025). https://doi.org/10.1038/s41467-025-67186-6

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Received: 10 April 2025

Accepted: 24 November 2025

Published: 08 December 2025

DOI: https://doi.org/10.1038/s41467-025-67186-6