Development of solid-state NMR methodology to study RNA and protein-RNA complexes

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This application is the extension of a grant with the same name, which has been funded by the DFG from 2014 to 2018. In the first funding period, we had three main goals: 1. Development of solid-state NMR (ssNMR) experiments for the sequential assignment of nucleotides in RNA; 2. Development of experiments to collect distance restraints and other relevant structural parameters by ssNMR; 3. Calculation of the RNA structure in an RNA-protein complex. Nearly all goals of the first funding period have been readily achieved. We have developed a straightforward, manageable strategy that uses easy-to-produce nucleotide-type selective-labeled RNAs and sensitive magnetization transfer schemes to obtain the structure of anRNA by ssNMR. In addition, we proved the applicability of our method to short stretches of labelled RNA in the context of large RNP complexes.In the work conducted in the first funding period, we used 13C detected experiments; the severe overlap of the NMR resonances typical for RNA and the low sensitivity of 3D experiments required using nucleotide type selective labeling to resolve overlaps in 2D experiments. This strategy is likely to reach its limits for RNA containing long canonical helices, where resonance overlap is more severe. Thus, the development of strategies that make 3–dimensional spectra feasible in the context of RNA and RNP complexes is indispensable to establish solid-state NMR as a valid tool for structural determination of either large RNAs or RNA as part of large particles. Following the great success of 1H detection in ssNMR of proteins, we explored the possibility of applying ultrafast MAS, 1H-detected solid-state NMR spectroscopy to RNA. Our choice to move towards 1H detection for RNA in ssNMR is motivated by to main goals: 1. Take advantage of the much longer life time of 13C at >100 kHz spinning rates to perform high-dimensionality experiments with high sensitivity; 2. Allow structural determination of RNA by ssNMR using submilligram quantities of material and only one sample with uniform labeling. The aim of this second funding period is to develop an experimental approach, based on 1H detection and fast MAS rates, to solve the structure of RNA by ssNMR.