FRET-guided modeling of nucleic acids

The functional diversity of RNA is encoded in their innate conformational heterogeneity. The combination of single-molecule spectroscopy and computational modeling offers new, attractive opportunities to map structural transitions within nucleic acid ensembles. Here, we describe a framework to harmonize single-molecule FRET measurements with molecular dynamics simulations and de novo structure prediction. Using either all-atom or implicit fluorophore modeling we recreate FRET experiments in silico , visualize the underlying structural dynamics and quantify the simulated reaction coordinates. Using multiple accessible-contact volumes (multi-ACV) as a post-hoc scoring method for fragment-assembly in Rosetta, we demonstrate that FRET effectively refines de novo RNA structure prediction without the need of explicit dye labeling. We benchmark our FRET-assisted modeling approach on double-labeled DNA strands and validate it against an intrinsically dynamic manganese(II)-binding riboswitch. We show that a FRET coordinate describing the assembly of a four-way junction allows our pipeline to recapitulate the global fold of the riboswitch with sub-helical accuracy to the crystal structure. We conclude that computational fluorescence spectroscopy facilitates the interpretability of dynamic structural ensembles and improves the mechanistic understanding of nucleic acid interactions.