Conformational dynamics at the pre-miR-377 Dicer site governs selective small-molecule recognition

Manigrasso, J.; mestdach, E.; quaia, R.; Dankova, D.; Bood, M.; Lemurell, M.; Geschwindner, S.; Frolov, A.; Disney, M.; Hogner, a.; Chi, c. N.; La Sala, G.

doi:10.64898/2026.02.11.705234

bioRxiv · 10.64898/2026.02.11.705234

Conformational dynamics at the pre-miR-377 Dicer site governs selective small-molecule recognition

Manigrasso, J.·mestdach, E.·quaia, R.·Dankova, D.·Bood, M.·Lemurell, M.·Geschwindner, S.·Frolov, A.·Disney, M.·Hogner, a.·Chi, c. N.·La Sala, G.

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Abstract

Small molecules that target precursor microRNAs (pre-miRNAs) offer a strategy to modulate microRNA biogenesis, but rational design is limited by the intrinsic conformational dynamics of RNA. Here, we combine site-specific nuclear magnetic resonance (NMR) spectroscopy with enhanced-sampling molecular dynamics (MD) simulations to define the conformational ensemble of pre-miR-377 and its recognition by the small-molecule binder C1. Our data reveals that the Dicer cleavage site contains a dynamic adenosine bulge in which U25 shuffles between A3 and A4 with a [~]9:1 population ratio, generating two interconverting RNA triple structures. NMR-informed MD and binding simulations show that C1 engages this dynamic region through a pathway-dependent mechanism that favors minor-groove entry, stabilizes intermediate, partially reorganized Dicer-site conformations, and redistributes the conformational ensemble away from processing-competent states. A conformationally locked mutant abolishes binding, indicating that recognition depends on bulge-enabled RNA dynamics rather than a single static structure. These results define the structural and dynamic basis of small-molecule recognition at the pre-miR-377 Dicer site and highlight the importance of RNA conformational ensembles in RNA-targeted drug discovery.

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Manigrasso, J., mestdach, E., quaia, R., Dankova, D., Bood, M., Lemurell, M., Geschwindner, S., Frolov, A., Disney, M., Hogner, a., Chi, c. N., La Sala, G.. 2026-02-12. Conformational dynamics at the pre-miR-377 Dicer site governs selective small-molecule recognition. https://doi.org/10.64898/2026.02.11.705234

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