Fig. 2: Viral-derived and engineered symmetric RNA molecules for structural stabilization of the SARS-CoV-2 N dimer.

A Genome organization of SARS-CoV-2 with boxes indicating different genes and empty rectangles indicating high-affinity binding RNA sequences. 5’UTR, 5’ untranslated regions; PS, packaging signal, and 3’UTR, 3’ untranslated regions. Representative NS-EM micrographs of three independent experiments of the purified RNA-free N dimer combined to secondary-structure based fragments of the viral PS (scale bar 100 nm). The predicted secondary structure of each RNA fragment (Vienna RNA fold server) is shown above the micrographs. The NS-EM reconstruction maps of the structurally homogeneous complexes are embedded in the corresponding micrographs. B Engineered 24-bp RNA formed by two copies of the high-affinity binding sequence found in PDB 7ACS connected by a 10-bp RNA linker (BLB-RNA). The EM-map of the dimeric N protein bound to the BLB-RNA using a low threshold to show the pseudo-two-fold symmetry is embedded in a representative NS-EM micrograph. C Cartography of the domains of N mapped in the EM reconstruction. D Mapping of cross-linked residues on 3D structure of N dimer. Above: crosslinked residues obtained by mass spectrometry (MS) from three independent experiments are depicted as arcs. Below: Three cross-linking reactions were carried out using the MS-cleavable cross-linker disuccinimidyl sulfoxide (DSSO) that bridges lysines, threonines, or serines with Cα-Cα distance range of 8–27 Å. The chemical structure of the cleavable cross-linker DSSO is shown (center). Representative MS spectra of cross-linked N peptides using DSSO at 5 (green), 10 (purple), and 50 (blue) times the estimated final protein concentration of 10 µM (center). The common residues across all the experiments are shown in the Venn diagram and the tables (right). Mapping (left) of the cross-linked residues in the 3D structure of SARS-CoV-2 N dimer obtained by EM common across all the experiments (black lines) or across at least two replicates (dashed lines) (Supplementary Fig. 5). In this 3D envelope, densities are assigned to the RNA-binding domain (RNA-BD), an intrinsically disordered region (IDR_central), and the dimerization domain (DD) of each monomer.