Vortrag auf dem Treffen der Deutschen Gesellschaft für Biophysik (DGfB e.V.) 2024, Leipzig

FRET-based integrative modeling for the characterization of an ribosomal RNA tertiary contact. Tertiary contact interactions between RNA GAAA tetraloops (TL) and their receptors play a crucial role in stabilizing ribosomal RNA folds and facilitating ribosomal maturation [1]. This study uses Förster-Resonance-Energy-Transfer (FRET)-based integrative modeling as an approach to investigate an RNA tertiary contact, comprising a Kissing-loop (KL) which acts as TL receptor [2]. We developed a FRET-assisted modeling pipeline to automate RNA structure prediction. The pipeline includes a knowledge-based modeling approach with PyMOL, MD simulations and the prediction of in silico FRET distributions with FRETraj [3] from all-atom dye and a fluorescence anisotropy weighted multiple accessible contact volume (mACV) simulations [4]. The simulations were compared to a cryo-EM structure and experimental in vitro single-molecule FRET data [5]. An unconstrained MD simulation showed a slight change in the KL-TL binding pattern compared to the cryo-EM structure, resulting in a mACV-derived FRET distribution that remarkably matches the FRET distribution of an in vitro experiment. However, all-atom dye simulations don’t represent the dye-RNA interactions leading to deviations in the in silico dynamic fluorescence anisotropy and FRET of the Cy3/5 dye pair. Our FRET-guided integrative modeling pipeline has the potential to enhance the understanding of RNA structures and their function, ultimately contributing to the development of more accurate RNA models based on experimental knowledge.