Elucidating RNA structural dynamics by means of smFRET and nsFCS on all time scales and building dynamic RNA structure models (RNAdyn)

• Applicant: Professor Dr. Richard Börner
• Subject Area: Biophysics ; Structural Biology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 537331625

The project focuses on the investigation of fast intrinsic domain movements of fluorescently labelled ribosomal ribonucleic acids (RNA) of baker's yeast. A sequence of ribosomal RNA containing a pseudoknot / kissing loop and its interaction with a GAAA tetraloop serves as a model system. This model system will be investigated by nanosecond-resolved correlation analysis (nsFCS) and multiparameter fluorescence detection (MFD) accompanied by pulsedinterleaved excitation (PIE) or alternated laser exciation (ALEX) based dye-sorted single molecule-resolved Förster resonance energy transfer (smFRET) experiments. Correlation analysis of time correlated single photon will allow us to obtain biomolecular motion patterns over theoretically 10 orders of magnitude in time (ps - h). We will investigate different time ranges that will give us information about the time regime of RNA domain movements (ps - ns possibly up to the μs-Bereich), s range), tertiary contact formations (μs-Bereich), s to seconds) as well as conformational and folding dynamics (ms - seconds). By simultaneously detecting two colour channels in FRET-FCS experiments, it is possible to map the anti-correlated change in photon count rates directly to the rate constant of a distance change in the molecule. By simultaneously splitting the measurement signal into two polarisation channels (s and p) of the two colour channels, the rate constant of a re-orientation of the dyes during the recorded motion patterns is also possible. The latter not only gives us insight into the freedom of movement of the dyes, an essential criterion for the quality of the distance information in the context of a FRET experiment, but also allows us to investigate the rotational speed of the RNA biomolecule as a whole or its labelled domains. The timeresolved distance information obtained in this way is to be used in a second step for the elucidation of RNA structures by pre-sorting socalled de novo structural models with regard to the distance information obtained from the FRET trajectory and thus drastically restricting the simulated structural ensemble (keyword: integrative hybrid modelling). Further, FRET-constraint MD simulations will help us to adapt the identified RNA structure to the experimental smFRET data, thus, enabeling truely experimenatlly validated solution structures of RNA biomolecules. Our aim is not only to improve the FRET-assisted structural models of RNA, but also to bring the static structural images obtained in this way to life, so to speak, with the velocity or rate information, and to enable realistic video sequences of molecular motion. This represents a milestone in the evaluation of biomolecular structures, e. g. in structure-based drug discovery and medical diagnostics.

• DFG Programme: Research Grants