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Fast (＞100 kHz) magic angle spinning solid-state NMR allows combining high-sensitive proton detection with the absence of an intrinsic molecular weight limit. Using this technique we observe for the first time narrow 1H RNA resonances and assign nucleotide spin systems with only 200 μg of uniformly 13C,15N-labelled RNA. [ABSTRACT FROM AUTHOR]

Sensitivity and specificity are both crucial for the efficient solid-state NMR structure determination of large biomolecules. We present an approach that features both advantages by site-specific enhancement of NMR spectroscopic signals from the protein-RNA binding site within a ribonucleoprotein (RNP) by dynamic nuclear polarization (DNP). This approach uses modern biochemical techniques for sparse isotope labeling and exploits the molecular dynamics of 13 C-labeled methyl groups exclusively present in the protein. These dynamics drive heteronuclear cross relaxation and thus allow specific hyperpolarization transfer across the biomolecular complex's interface. For the example of the L7Ae protein in complex with a 26mer guide RNA minimal construct from the box C/D complex in archaea, we demonstrate that a single methyl-nucleotide contact is responsible for most of the polarization transfer to the RNA, and that this specific transfer can be used to boost both NMR spectral sensitivity and specificity by DNP., (© 2022 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

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., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

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 13 C- and 15 N-detected experiments and novel 1 H-detected methods, identify optimal regimes for RNA studies by ssNMR, and provide our view on future ssNMR studies of RNA in large RNP complexes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Aguion and Marchanka.)

Solid-state NMR (ssNMR) is applicable to high molecular-weight (MW) protein assemblies in a non-amorphous precipitate. The technique yields atomic resolution structural information on both soluble and insoluble particles without limitations of MW or requirement of crystals. Herein, we propose and demonstrate an approach that yields the structure of protein-RNA complexes (RNP) solely from ssNMR data. Instead of using low-sensitivity magnetization transfer steps between heteronuclei of the protein and the RNA, we measure paramagnetic relaxation enhancement effects elicited on the RNA by a paramagnetic tag coupled to the protein. We demonstrate that this data, together with chemical-shift-perturbation data, yields an accurate structure of an RNP complex, starting from the bound structures of its components. The possibility of characterizing protein-RNA interactions by ssNMR may enable applications to large RNP complexes, whose structures are not accessible by other methods., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

RNA structure is essential to understand RNA function and regulation in cellular processes. RNA acts either in isolation or as part of a complex with proteins. Both isolated and protein-complexed RNA represent a challenge for structural biology, due to the complexity of its conformational space and intrinsic dynamics. NMR, with its capability to cope with dynamic structures, is the optimal technique to study RNA conformation, ideally in solution. However, RNA-protein complexes are often very large, which limits the application of solution-state NMR. In this chapter we describe the methodology that we developed to determine the structure of RNA by solid-state NMR. Solid-state NMR is not limited by large molecular weights and can be optimally applied to study both the RNA and the protein components of large RNA-protein complexes. We review the methods for resonance assignments, collection of RNA-specific distance restraints, as well as detection of protein-RNA interfaces., (© 2019 Elsevier Inc. All rights reserved.)

Intranucleotide resonance of the 26mer box C/D RNA in complex with the L7Ae protein were assigned by solid-state NMR (ssNMR; see picture) spectroscopy. This investigation opens the way for studying RNA in large protein-RNA complexes by ssNMR spectroscopy., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)