NMR-based developments to study RNA refolding and RNA ligand binding/druggability

• Applicant: Professor Dr. Harald Schwalbe
• Subject Area: Physical Chemistry of Molecules, Liquids and Interfaces, Biophysical Chemistry ; Structural Biology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 560722850

Nuclear magnetic resonance (NMR) plays an essential in the structural biology of RNAs. RNA’s conformational spaces can only be faithfully described by ensembles of fluctuating structures. This often prevents crystallization attempts on RNAs, and makes the structuro-kinetic, atomic-level insight afforded by solution phase NMR essential in understanding these systems. To fully exploit NMR’s potential in RNA structural biology, it is essential to overcome its signal-to-noise limitations while maximizing site resolution. This proposal addresses these aspects with improvements in NMR methodologies specifically tailored to the study of RNAs. With the aid of these techniques, which we expect to improve by 2-3 orders-of-magnitude the sensitivity of contemporary methods, we plan to exploit the ultrahigh field NMR infrastructure installed both in Rehovot and Frankfurt to (i) detect ligand binding to RNA by both ligand-based and RNA-target-based screening experiments, and (ii) characterize the kinetics of RNA refolding induced by ligand binding at a nucleotide-level resolution. The proposed methodologies have in common a reliance on the rapid exchange that hydrogen sites in RNAs –in particular 2’-OH in the ribose ring, and NH, NH2 in the nucleobases– undergo with the solvent (water). Under these conditions, (i) new frequency-selective experiments exploiting the transfer of the large water magnetization reservoir recently demonstrated in Frydman/Schwalbe collaborations will be fine-tuned for optimized experiments that detect structural and dynamic changes as well as ligand binding by changes of water accessibility to RNAs; and (ii) dissolution dynamic nuclear polarization (DNP) experiments based on the injection of hyperpolarized water and ligands available at Weizmann will be used to characterize ligand-induced RNA refoldings. Both methods will be benchmarked on (i) an aptamer domain of a guanine-sensing tandem riboswitch and on the frameshifting element of SARS-CoV-2 which controls highly relevant RNA refolding events and is an active target for NMR-guided development of antiviral inhibitors. The outcome of the project here thus directly fuels into the on-going development of antiviral inhibitors in the Schwalbe lab. The proposed project brings together the unique expertise of the two groups in NMR of RNA (Schwalbe) and emerging forms of multidimensional and DNP dissolution-based methods (Frydman) –with unique access to NMR and DNP facilities and with a proven track record of collaboration in the field.

• DFG Programme: Research Grants
• International Connection: Israel
• Partner Organisation: The Israel Science Foundation
• Cooperation Partner: Professor Dr. Lucio Frydman