Identification of RNA Base Pairs and Complete Assignment of Nucleobase Resonances by Proton-Detected Solid-State NMR Spectroscopy at 100†kHz MAS.

Knowledge of RNA structure, either in isolation or in complex, is fundamental to understand the mechanism of cellular processes. Solid-state NMR (ssNMR) is applicable to high molecular-weight complexes and does not require crystallization; thus, it is well-suited to study RNA as part of large multicomponent assemblies. Recently, we solved the first structures of both RNA and an RNA-protein complex by ssNMR using conventional 13 C- and 15 N-detection. This approach is limited by the severe overlap of the RNA peaks together with the low sensitivity of multidimensional experiments. Here, we overcome the limitations in sensitivity and resolution by using 1 H-detection at fast MAS rates. We develop experiments that allow the identification of complete nucleobase spin-systems together with their site-specific base pair pattern using sub-milligram quantities of one uniformly labelled RNA sample. These experiments provide rapid access to RNA secondary structure by ssNMR in protein-RNA complexes of any size.

Nucleic acid-protein interfaces studied by MAS solid-state NMR spectroscopy.

Solid-state NMR (ssNMR) has become a well-established technique to study large and insoluble protein assemblies. However, its application to nucleic acid-protein complexes has remained scarce, mainly due to the challenges presented by overlapping nucleic acid signals. In the past decade, several efforts have led to the first structure determination of an RNA molecule by ssNMR. With the establishment of these tools, it has become possible to address the problem of structure determination of nucleic acid-protein complexes by ssNMR. Here we review first and more recent ssNMR methodologies that study nucleic acid-protein interfaces by means of chemical shift and peak intensity perturbations, direct distance measurements and paramagnetic effects. At the end, we review the first structure of an RNA-protein complex that has been determined from ssNMR-derived intermolecular restraints.

Strategies for RNA Resonance Assignment by 13C/15N- and 1H-Detected Solid-State NMR Spectroscopy.

Magic angle spinning (MAS) solid-state NMR (ssNMR) is an established tool that can be applied to non-soluble or non-crystalline biomolecules of any size or complexity. The ssNMR method advances rapidly due to technical improvements and the development of advanced isotope labeling schemes. While ssNMR has shown significant progress in structural studies of proteins, the number of RNA studies remains limited due to ssNMR methodology that is still underdeveloped. Resonance assignment is the most critical and limiting step in the structure determination protocol that defines the feasibility of NMR studies. In this review, we summarize the recent progress in RNA resonance assignment methods and approaches for secondary structure determination by ssNMR. We critically discuss advantages and limitations of conventional 13C- and 15N-detected experiments and novel 1H-detected methods, identify optimal regimes for RNA studies by ssNMR, and provide our view on future ssNMR studies of RNA in large RNP complexes.