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The SARS-CoV-2 (SCoV-2) virus is the causative agent of the ongoing COVID-19 pandemic. It contains a positive sense single-stranded RNA genome and belongs to the genus of Betacoronaviruses. The 5′- and 3′-genomic ends of the 30 kb SCoV-2 genome are potential antiviral drug targets. Major parts of these sequences are highly conserved among Betacoronaviruses and contain cis-acting RNA elements that affect RNA translation and replication. The 31 nucleotide (nt) long highly conserved stem-loop 5a (SL5a) is located within the 5′-untranslated region (5′-UTR) important for viral replication. SL5a features a U-rich asymmetric bulge and is capped with a 5′-UUUCGU-3′ hexaloop, which is also found in stem-loop 5b (SL5b). We herein report the extensive 1H, 13C and 15N resonance assignment of SL5a as basis for in-depth structural studies by solution NMR spectroscopy.
The stem-loop (SL1) is the 5'-terminal structural element within the single-stranded SARS-CoV-2 RNA genome. It is formed by nucleotides 7–33 and consists of two short helical segments interrupted by an asymmetric internal loop. This architecture is conserved among Betacoronaviruses. SL1 is present in genomic SARS-CoV-2 RNA as well as in all subgenomic mRNA species produced by the virus during replication, thus representing a ubiquitous cis-regulatory RNA with potential functions at all stages of the viral life cycle. We present here the 1H, 13C and 15N chemical shift assignment of the 29 nucleotides-RNA construct 5_SL1, which denotes the native 27mer SL1 stabilized by an additional terminal G-C base-pair.
We report here the nuclear magnetic resonance 19F screening of 14 RNA targets with different secondary and tertiary structure to systematically assess the druggability of RNAs. Our RNA targets include representative bacterial riboswitches that naturally bind with nanomolar affinity and high specificity to cellular metabolites of low molecular weight. Based on counter-screens against five DNAs and five proteins, we can show that RNA can be specifically targeted. To demonstrate the quality of the initial fragment library that has been designed for easy follow-up chemistry, we further show how to increase binding affinity from an initial fragment hit by chemistry that links the identified fragment to the intercalator acridine. Thus, we achieve low-micromolar binding affinity without losing binding specificity between two different terminator structures.
We present here a set of 13C-direct detected NMR experiments to facilitate the resonance assignment of RNA oligonucleotides. Three experiments have been developed: (1) the (H)CC-TOCSY-experiment utilizing a virtual decoupling scheme to assign the intraresidual ribose 13C-spins, (2) the (H)CPC-experiment that correlates each phosphorus with the C40 nuclei of adjacent nucleotides via J(C,P) couplings and (3) the (H)CPC-CCH-TOCSY-experiment that correlates the phosphorus nuclei with the respective C10,H10 ribose signals. The experiments were applied to two RNA hairpin structures. The current set of 13C-direct detected experiments allows direct and unambiguous assignment of the majority of the hetero nuclei and the identification of the individual ribose moieties following their sequential assignment. Thus, 13C-direct detected NMR methods constitute useful complements to the conventional 1H-detected approach for the resonance assignment of oligonucleotides that is often hindered by the limited chemical shift dispersion. The developed methods can also be applied to large deuterated RNAs. Keywords: NMR spectroscopy , Direct carbon , detection , RNA