FRET-guided integrative modelling: A workflow for RNA tertiary contact prediction.
Understanding the complex biomolecular mechanisms underlying long-range RNA tertiary contacts [1] is crucial for advancing RNA-based therapeutics and drug discovery applications. Although current state-of-the-art structure prediction methods like RNAfold [2] for secondary structures and de novo modelling such as RNAcomposer [3] and Rosetta farfar2 [4] yield remarkable results, they struggle to compose binding events of spatially separated secondary structure elements, i.e., the formation of RNA tertiary contacts. Here, we introduce a streamlined workflow, which integrates de novo structure prediction through Rosetta paired with MD simulation using GROMACS followed by in silico FRET predictions with FRETraj [5] as a FRET-guided integrative modelling approach to capture RNA structural dynamics and to gain RNA folding pathways [6-8]. By integrating smFRET experimental parameters, e.g., dye quantum yield, burst size distribution and detection efficiency, we not only successfully model an RNA tertiary contact originated in the 25S ribosomal RNA of baker’s yeast [9], but yield an exceptionally agreement with the experimental derived smFRET distributions. We conclude that computational fluorescence spectroscopy facilitates the interpretability of dynamic structural ensembles and improves the mechanistic understanding of nucleic acid interactions. [1] P. Nissen et al., PNAS, 98, 4899–4903 (2001) doi.org/10.1073/pnas.081082398. [2] R. Lorenz et al., AMB, 6, 26 (2011) doi.org/10.1186/1748-7188-6-26. [3] M. Biesiada et al., Methods, 103, 120–127 (2016) doi.org/10.1016/j.ymeth.2016.03.010. [4] A. M. Watkins et al., Structure, 28, 963-976.e6 (2020) doi.org/10.1016/j.str.2020.05.011. [5] F. D. Steffen et al., Bioinformatics, 37, 21, 3953-3955 (2021) doi.org/10.1093/bioinformatics/btab615. [6] F. Erichson et al., Hochschule Mittweida, 2, 230–233 (2021) doi.org/10.48446/opus-12283. [7] M. Dimura et al., Curr. Opin. Struct. Biol., 40, 163–185 (2016) doi.org/10.1016/j.sbi.2016.11.012. [8] F. D. Steffen et al., Biorxiv, (2023) doi.org/10.1101/2023.08.07.552238. [9] S. Gerhardy et al., Nat. Commun., 12, 4696 (2021) doi.org/10.1038/s41467-021-24964-2.