Automated and assisted RNA resonance assignment using NMR chemical shift statistics

The three-dimensional structure determination of RNAs by NMR spectroscopy relies on chemical shift assignment, which still constitutes a bottleneck. In order to develop more efficient assignment strategies, we analysed relationships between sequence and 1 H and 13 C chemical shifts. Statistics of resonances from regularly Watson– Crick base-paired RNA revealed highly characteristic chemical shift clusters. We developed two approaches using these statistics for chemical shift assignment of double-stranded RNA (dsRNA): a manual approach that yields starting points for resonance assignment and simplifies decision trees and an automated approach based on the recently introduced automated resonance assignment algorithm FLYA. Both strategies require only unlabeled RNAs and three 2D spectra for assigning the H2/C2, H5/C5, H6/C6, H8/C8 and H1 0 /C1 0 chemical shifts. The manual approach proved to be efficient and robust when applied to the experimental data of RNAs with a size between 20nt and 42nt. The more advanced automated assignment approach was successfully applied to four stemloop RNAs and a 42nt siRNA, assigning 92–100% of the resonances from dsRNA regions correctly. This is the first automated approach for chemical shift assignment of non-exchangeable protons of RNA and their corresponding 13 C resonances,