A hybrid structure determination approach to investigate the druggability of the nucleocapsid protein of SARS-CoV-2.

The pandemic caused by SARS-CoV-2 has called for concerted efforts to generate new insights into the biology of betacoronaviruses to inform drug screening and development. Here, we establish a workflow to determine the RNA recognition and druggability of the nucleocapsid N-protein of SARS-CoV-2, a highly abundant protein crucial for the viral life cycle. We use a synergistic method that combines NMR spectroscopy and protein-RNA cross-linking coupled to mass spectrometry to quickly determine the RNA binding of two RNA recognition domains of the N-protein. Finally, we explore the druggability of these domains by performing an NMR fragment screening. This workflow identified small molecule chemotypes that bind to RNA binding interfaces and that have promising properties for further fragment expansion and drug development.

1H, 13C and 15N chemical shift assignment of the stem-loops 5b + c from the 5'-UTR of SARS-CoV-2.

The ongoing pandemic of the respiratory disease COVID-19 is caused by the SARS-CoV-2 (SCoV2) virus. SCoV2 is a member of the Betacoronavirus genus. The 30 kb positive sense, single stranded RNA genome of SCoV2 features 5'- and 3'-genomic ends that are highly conserved among Betacoronaviruses. These genomic ends contain structured cis-acting RNA elements, which are involved in the regulation of viral replication and translation. Structural information about these potential antiviral drug targets supports the development of novel classes of therapeutics against COVID-19. The highly conserved branched stem-loop 5 (SL5) found within the 5'-untranslated region (5'-UTR) consists of a basal stem and three stem-loops, namely SL5a, SL5b and SL5c. Both, SL5a and SL5b feature a 5'-UUUCGU-3' hexaloop that is also found among Alphacoronaviruses. Here, we report the extensive 1H, 13C and 15N resonance assignment of the 37 nucleotides (nts) long sequence spanning SL5b and SL5c (SL5b + c), as basis for further in-depth structural studies by solution NMR spectroscopy.

Cover Feature: The Extended Hadamard Transform: Sensitivity-Enhanced NMR Experiments Among Labile and Non-Labile 1Hs of SARS-CoV-2-derived RNAs (ChemPhysChem 4/2022)

The Cover Feature illustrates how artifact-free 2D NOE correlations between labile protons can be obtained from an extended Hadamard encoding/decoding matrix, which supersedes problems in conventional Hadamard schemes. The sensitivity-enhancing abilities of extended Hadamard encoding operating in conjunction with solvent repolarization mechanisms are demonstrated on GHz NMR studies on SARS-CoV-2 RNA fragments. More information can be found in the Article by Lucio Frydman and co-workers.

The Extended Hadamard Transform: Sensitivity-Enhanced NMR Experiments Among Labile and Non-Labile 1 Hs of SARS-CoV-2-derived RNAs.

Hadamard encoded saturation transfer can significantly improve the efficiency of NOE-based NMR correlations from labile protons in proteins, glycans and RNAs, increasing the sensitivity of cross-peaks by an order of magnitude and shortening experimental times by ≥100-fold. These schemes, however, fail when tackling correlations within a pool of labile protons - for instance imino-imino correlations in RNAs or amide-amide correlations in proteins. Here we analyze the origin of the artifacts appearing in these experiments and propose a way to obtain artifact-free correlations both within the labile pool as well as between labile and non-labile 1 Hs, while still enjoying the gains arising from Hadamard encoding and solvent repolarizations. The principles required for implementing what we define as the extended Hadamard scheme are derived, and its clean, artifact-free, sensitivity-enhancing performance is demonstrated on RNA fragments derived from the SARS-CoV-2 genome. Sensitivity gains per unit time approaching an order of magnitude are then achieved in both imino-imino and imino-amino/aromatic protons 2D correlations; similar artifact-free sensitivity gains can be observed when carrying out extended Hadamard encodings of 3D NOESY/HSQC-type experiments. The resulting spectra reveal significantly more correlations than their conventionally acquired counterparts, which can support the spectral assignment and secondary structure determination of structured RNA elements.

3D Heteronuclear Magnetization Transfers for the Establishment of Secondary Structures in SARS-CoV-2-Derived RNAs.

Multidimensional NOESY experiments targeting correlations between exchangeable imino and amino protons provide valuable information about base pairing in nucleic acids. It has been recently shown that the sensitivity of homonuclear correlations involving RNA's labile imino protons can be significantly enhanced, by exploiting the repolarization brought about by solvent exchanges. Homonuclear correlations, however, are of limited spectral resolution, and usually incapable of tackling relatively large homopolymers with repeating structures like RNAs. This study presents a heteronuclear-resolved version of those NOESY experiments, in which magnetization transfers between the aqueous solvent and the nucleic acid protons are controlled by selecting specific chemical shift combinations of a coupled 1H-15N spin pair. This selective control effectively leads to a pseudo-3D version of HSQC-NOESY, but with cross-peaks enhanced by ∼2-5× as compared with conventional 2D NOESY counterparts. The enhanced signal sensitivity as well as access to both 15N-1H and 1H-1H NOESY dimensions can greatly facilitate RNA assignments and secondary structure determinations, as demonstrated here with the analysis of genome fragments derived from the SARS-CoV-2 virus.

Magnetization Transfer to Enhance NOE Cross-Peaks among Labile Protons: Applications to Imino-Imino Sequential Walks in SARS-CoV-2-Derived RNAs.

2D NOESY plays a central role in structural NMR spectroscopy. We have recently discussed methods that rely on solvent-driven exchanges to enhance NOE correlations between exchangeable and non-exchangeable protons in nucleic acids. Such methods, however, fail when trying to establish connectivities within pools of labile protons. This study introduces an alternative that also enhances NOEs between such labile sites, based on encoding a priori selected peaks by selective saturations. The resulting selective magnetization transfer (SMT) experiment proves particularly useful for enhancing the imino-imino cross-peaks in RNAs, which is a first step in the NMR resolution of these structures. The origins of these enhancements are discussed, and their potential is demonstrated on RNA fragments derived from the genome of SARS-CoV-2, recorded with better sensitivity and an order of magnitude faster than conventional 2D counterparts.

Magnetization Transfer to Enhance NOE Cross-Peaks among Labile Protons: Applications to Imino-Imino Sequential Walks in SARS-CoV-2-Derived RNAs.

2D NOESY plays a central role in structural NMR spectroscopy. We have recently discussed methods that rely on solvent-driven exchanges to enhance NOE correlations between exchangeable and non-exchangeable protons in nucleic acids. Such methods, however, fail when trying to establish connectivities within pools of labile protons. This study introduces an alternative that also enhances NOEs between such labile sites, based on encoding a priori selected peaks by selective saturations. The resulting selective magnetization transfer (SMT) experiment proves particularly useful for enhancing the imino-imino cross-peaks in RNAs, which is a first step in the NMR resolution of these structures. The origins of these enhancements are discussed, and their potential is demonstrated on RNA fragments derived from the genome of SARS-CoV-2, recorded with better sensitivity and an order of magnitude faster than conventional 2D counterparts.

Secondary structure determination of conserved SARS-CoV-2 RNA elements by NMR spectroscopy.

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.