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 13C‐ and 15N‐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 1H‐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.

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.

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Die Aufklärung von RNAS trukturen, entweder in Isolation oder im Komplex, ist essentiell, um die Mechanismen zellulärer Prozesse zu verstehen. Festkçrper-NMR ist auf Komplexem it hohem Molekulargewicht anwendbar und bençtigt keine Kristallisation;Die Methode ist daher gut geeignet um RNAals Teil von großen mehrkomponentigen Komplexen zu untersuchen. Kürzlich konnten wir die ersten Strukturen von RNAu nd einem RNA-Protein Komplex mittels Festkçrper-NMR durch die Anwendung von konventioneller 13 C-und 15 N-Detektion lçsen. Dieser Ansatz ist durch diverse RNA Peak-Überlappungen und die niedrige Intensitätv on multidimensionalen Experimenten limitiert. In dieser Arbeit überwinden wir die Limitationen in Sensitivitätu nd Auflçsung durch die Anwendung von 1 H-Detektion bei schnellenM AS Geschwindigkeiten. Wir entwickeln Experimente,d ie die Identifizierung von vollständigen Spinsystemen der Nukleobasen zusammen mit derer Nukleotid-spezifischer Basenpaarung ermçglichen. Diese Experimente bençtigen weniger als ein Milligramm einer uniform Isotopen-markierten RNA Probe und erlauben die schnelle Bestimmung der RNAS ekundärstruktur durch Festkçrper-NMR in Protein-RNA Komplexen jedweder Grçße.

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 13C- and 15N-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 1H-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.