BPS2026 – Linking RNA sequence to structure and dynamics through high-throughput single-molecule FRET and sequencing (smFAS)

Single-molecule Förster resonance energy transfer (smFRET) has transformed our understanding of RNA folding and RNA-protein interactions by directly resolving conformational heterogeneity and dynamics. smFRET assays are traditionally restricted to one sequence at a time, which limits their application to large sequence spaces. To overcome this limitation, we are developing and applying single-molecule FRET and sequencing (smFAS), an integrated approach that links conformational dynamics to RNA sequence identity in a massively parallel manner. Yeast Ubc4 pre-mRNA was utilized as a model substrate to demonstrate how the branchpoint sequence (BPS) impacts early spliceosome assembly. Using smFRET, we observed fluctuations in the proximity of the branchpoint and 5′ splice site, with wild-type pre-mRNA slightly favoring an undocked (lower FRET) ensemble. Splicing-competent conformations are stabilized by binding branchpoint binding protein (BBP), which we demonstrate shifts equilibrium toward a docked, higher FRET state. Mutational analyses revealed that single-nucleotide substitutions within the BPS adjust docking free energies over a significant range, illustrating how conformational diversity is encoded by pre-mRNA sequence. Even after protein washout, conformational bias induced by BBP persists, suggesting conformational memory or slow dissociation kinetics. The smFAS platform has also been applied to the preQ 1 riboswitch. This method revealed ligand-dependent pseudoknot docking in the wild-type RNA, validating smFAS as a generalizable platform for RNA structural biology. In summary, smFAS is a powerful tool for mapping RNA sequence-structure-function landscapes at single-molecule resolution and high throughput, with implications for understanding splicing regulation, improving RNA structure prediction algorithms, and identifying disease-related sequence effects.