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

Abstract

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.

SARS-CoV-2 highly conserved s2m element dimerizes via a kissing complex and interacts with host miRNA-1307-3p

“…We employed one-dimensional 1 H NMR spectroscopy to analyze the secondary structures of these mutants to determine if they resemble the SARS-CoV s2m or SARS-CoV-2 s2m secondary structure. The SARS-CoV-2 s2m imino proton resonance region ( Figure 6A, top spectrum) matched that of its previously determined secondary structure, allowing us to assign its resonances (12). It should be noted, however, that our analysis focused on a synthesized 41-nucleotide sequence corresponding to the SARS-CoV-2 s2m element, whereas the previous characterization included two additional G-C base pairs introduced in the lower stem for transcriptional purposes.…”

“…These elements include three stem-loops (SL1, SL2, and SL3) within the 5' UTR, as well as a bulged stem-loop (BSL), pseudoknot (PK) stem-loop, and hypervariable region (HVR) within the 3' UTR. Recent structural studies utilizing NMR spectroscopy, SHAPE mutational profiling, and various other characterization metrologies have confirmed the conservation of these elements within the SARS-CoV-2 genome (12)(13)(14). Moreover, a recent investigation of the short-and long-range interactions within the SARS-CoV-2 genome suggests these elements additionally function in genome cyclization and interactions with the frame-shifting elements of the gRNA ORF regions, indicating potential mechanistic roles in discontinuous transcription and viral protein translation events as well (15).…”

“…From the proposed SARS-CoV-2 s2m stem-loop structure, a 4-nt palindrome GUAC was observed within the loop, suggesting a possible site for the formation of a homodimeric kissing complex that could be implicated in genome dimerization ( Figure 2) (12). To investigate this proposed interaction, nondenaturing gel electrophoresis studies were carried out both in the presence and absence of Mg 2+ ions (Figure 3).…”

“…According to the X-ray crystal structure of SARS-CoV s2m, U5 is located in the lower stem and base-paired to G37, whereas G31 is located in the middle stem, base-paired with C12 ( Figure 5A) (18). The secondary structure of the SARS-CoV-2 s2m element was recently solved by NMR spectroscopy and is conformationally different from that of SARS-CoV s2m (12). These conformational variations were additionally confirmed by our 1.5 µs molecular dynamic simulations (manuscript submitted to Biophysical Journal, Special Issue: Biophysicists Address COVID-19 Challenges).…”

“…Characterization of the s2m element mutant constructs and their dimerization properties. One-dimensional 1 H NMR spectroscopy experiments were performed for all four s2m constructs in the presence of 0 mM MgCl2 (A), with resonances being assigned within the SARS-CoV-2 s2m spectrum based on the previously characterized secondary structure (12). All four s2m sequences were additionally incubated in the presence of 1 and 10 mM MgCl2, followed by electrophoresing in TBE and TBM nondenaturing gels (B).…”