Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

63-POS Board 31 VISUALISING TARGET RNA RECOGNITION IN TYPE III-B CRISPR SYSTEMS BY SMFRET Yu Sun ; Malcolm White 1 ; Carlos Penedo 2 ; 1 University of St Andrews, School of Biology, Biomedical Sciences Research Complex, St Andrews, United Kingdom 2 University of St Andrews, School of Physics and Astronomy, Centre for Biophotonics, Biomedical Sciences Research Complex, St Andrews, United Kingdom Type III CRISPR-Cas systems defend against invading RNA by coupling target recognition to RNA cleavage and cyclic oligoadenylate signalling. Although the molecular mechanism of these systems has been characterised, the dynamic mechanisms governing target RNA recognition and cleavage remain poorly understood. Here, we combine fluorescence anisotropy (FA) and single molecule fluorescence resonance energy transfer (smFRET) to investigate the interactions between Type III-B Cmr complexes and their RNA substrates.FA measurements reveal exceptionally slow dissociation kinetics for both the BfragCmr (Bacteroides fragilis) and VmeCmr (Vibrio metoecus) complexes, with preferential retention of the 5' cleavage product following RNA cleavage. smFRET further shows that free target RNA exists in multiple dynamically exchanging conformational states, whereas binding by the Cmr complex stabilises the RNA in predominantly low-FRET conformations, consistent with a more extended structure. These observations suggest that Cmr binding constrains the conformational landscape of the target RNA before or during cleavage, revealing dynamic intermediates that are inaccessible to conventional ensemble measurements. Together, our results provide a dynamic view of RNA recognition by Type III-B CRISPR-Cas systems and demonstrate how single-molecule approaches can uncover conformational mechanisms underlying target binding, cleavage, and product retention.

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