RNA studies by solid state NMR
With the study of large RNP complexes, we entered a new field to us: solid-state (ss) NMR. ssNMR is particularly suitable for large complexes, as the line-width is independent of the size of the molecule. We believe that there is an as yet unexplored potential of ssNMR spectroscopy in the investigation of RNP complexes. Pioneering the application of ssNMR to study RNA-protein interactions, we have proposed novel methods to detect atomic contacts at the RNA-protein interfaces (1,2). In addition, we developed technologies to assign RNA resonances in solid-state NMR, which is a prerequisite for structural determination of the nucleic acid components of RNP complexes ().
Recently, we reached a milestone in structure biology of RNA by solving the first de novo structure of RNA by ssNMR (Figure 1). We demonstrated that RNA structure is accessible at high-resolution by ssNMR using a few, easy to prepare, nucleotide-type selectively labeled samples. The methodology can be combined with segmental RNA-labelling to determine the structure of RNA stretches in high molecular-weight cellular machines and thus allows mechanistic studies of yet inaccessible RNA-protein complexes.
Figure 1. Left: Schematics of the ssNMR experiments used for resonance assignment and structure determination of the RNA by ssNMR. Right: Three-dimensional structure of the 26mer Box C/D RNA bound to the protein L7Ae (protein not shown), determined by ssNMR restraints (green, K-turn region; blue, NC- and C-stems; gray, loop).
1. Asami, S., Rakwalska-Bange, M., Carlomagno, T. & Reif, B. Protein-RNA interfaces probed by 1H-detected MAS solid-state NMR spectroscopy. Angew. Chem. Int. Ed. Engl. 52, 2345–9 (2013).
2. Jehle, S. et al. Intermolecular protein-RNA interactions revealed by 2D 31P-15N magic angle spinning solid-state NMR spectroscopy. J. Am. Chem. Soc. 132, 3842–6 (2010).
3. Marchanka, A., Simon, B. & Carlomagno, T. A suite of solid-state NMR experiments for RNA intranucleotide resonance assignment in a 21 kDa protein-RNA complex. Angew. Chem. Int. Ed. Engl. 52, 9996–10001 (2013).
4. Marchanka, A., Simon, B., Althoff-Ospelt, G. & Carlomagno, T. RNA structure determination by solid-state NMR spectroscopy. Nat. Commun. 6, 7024 (2015).