The current pandemic situation caused by the Betacoronavirus SARS-CoV-2 (SCoV2) highlights the need for coordinated research to combat COVID-19. A particularly important aspect is the development of medication. In addition to viral proteins, structured RNA elements represent a potent alternative as drug targets. The search for drugs that target RNA requires their high-resolution structural characterization. Using nuclear magnetic resonance (NMR) spectroscopy, a worldwide consortium of NMR researchers aims to characterize potential RNA drug targets of SCoV2. Here, we report the characterization of 15 conserved RNA elements located at the 5′ end, the ribosomal frameshift segment and the 3′-untranslated region (3′-UTR) of the SCoV2 genome, their large-scale production and NMR-based secondary structure determination. The NMR data are corroborated with secondary structure probing by DMS footprinting experiments. The close agreement of NMR secondary structure determination of isolated RNA elements with DMS footprinting and NMR performed on larger RNA regions shows that the secondary structure elements fold independently. The NMR data reported here provide the basis for NMR investigations of RNA function, RNA interactions with viral and host proteins and screening campaigns to identify potential RNA binders for pharmaceutical intervention.

Dynamic nuclear polarization (DNP) of a biomolecule tagged with a polarizing agent has the potential to not only increase NMR sensitivity but also to provide spectroscopy specificity towards the tagging...

Paramagnetic relaxation enhancement (PRE) is a versatile tool for NMR spectroscopic structural and kinetic studies in biological macromolecules. Here, we compare the quality of PRE data derived from two spin labels with markedly different dynamic properties for large RNAs using the I-A riboswitch aptamer domain (78 nt) from Mesoplamsa florum as model system. We designed two I-A aptamer constructs that were spin-labeled by noncovalent hybridization of short spin-labeled oligomer fragments. As an example of a flexible spin label, U-TEMPO was incorporated into the 3' terminal end of helix P1 while, the recently developed rigid spin-label Çm was incorporated in the 5' terminal end of helix P1. We determined PRE rates obtained from aromaticC bound proton intensities and compared these rates to PREs derived from imino proton intensities in this sizeable RNA (~78 nt). PRE restraints derived from both imino and aromatic protons yielded similar data quality, and hence can both be reliably used for PRE determination. For NMR, the data quality derived from the rigid spin label Çm is slightly better than the data quality for the flexible TEMPO as judged by comparison of the structural agreement with the I-A aptamer crystal structure (3SKI).

BACKGROUND: Seven dioxaborole compounds are investigated in this study. Structural and spectral characterizations are done at the M062X/6-31+G(d,p) level in water. Active sites of these compounds are determined by contour plots of frontier molecular orbital and molecular electrostatic potential (MEP) maps. Electrophilic and nucleophilic attack regions are determined. Since SARS-CoV-2 is a worldwide health problem, antiviral properties of studied boron-containing compounds are investigated by molecular docking calculations. In addition to these calculations, MM/PSBA calculations are performed. RESULTS AND CONCLUSION: It is found that the studied boron compounds can be good drug candidates against the main protease of SARS-CoV-2, while the best of them is 4,6-di-tert-butyl-2-(4-methoxyphenyl) benzo[d][1,3,2] dioxaborole (B2)

Funding for open access charge: DFG. This change does not affect the results, discussion and conclusions presented in the article. The published article has been updated.

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The ribosomal S1 protein (rS1) is indispensable for translation initiation in Gram-negative bacteria. rS1 is a multidomain protein that acts as an RNA chaperone and ensures that mRNAs can bind the ribosome in a single-stranded conformation, which could be related to fast recognition. Although many ribosome structures were solved in recent years, a high-resolution structure of a two-domain mRNA-binding competent rS1 construct is not yet available. Here, we present the NMR solution structure of the minimal mRNA-binding fragment of Vibrio Vulnificus rS1 containing the domains D3 and D4. Both domains are homologues and adapt an oligonucleotide-binding fold (OB fold) motif. NMR titration experiments reveal that recognition of miscellaneous mRNAs occurs via a continuous interaction surface to one side of these structurally linked domains. Using a novel paramagnetic relaxation enhancement (PRE) approach and exploring different spin-labeling positions within RNA, we were able to track the location and determine the orientation of the RNA in the rS1–D34 bound form. Our investigations show that paramagnetically labeled RNAs, spiked into unmodified RNA, can be used as a molecular ruler to provide structural information on protein-RNA complexes. The dynamic interaction occurs on a defined binding groove spanning both domains with identical β2-β3-β5 interfaces. Evidently, the 3′-ends of the cis-acting RNAs are positioned in the direction of the N-terminus of the rS1 protein, thus towards the 30S binding site and adopt a conformation required for translation initiation.

NMR spectroscopy plays a pivotal role for drug discovery since it can quantify interactions between ligands and molecular targets including proteins, DNA and RNA. Therefore, NMR methods can be used to gain structural insight into ligand-target complexes or to identify binding of ligands. However, its usage is limited due to factors like low sensibility, high expenditure of time or the necessity for isotope labelling. This chapter focuses on paramagnetic NMR methods and how they can overcome, at least partially, those limitations. Here, both methods for structural characterisation of ligand-target complexes and screening methods will be discussed in comparison to the diamagnetic alternative approaches, including methods based on paramagnetic relaxation enhancement, pseudo contact shift or hyperpolarisation.