Inhibition of SARS-CoV-2 nucleocapsid protein–RNA interaction by guanosine oligomeric RNA

Fig. 1. Inhibition of the NTD–RNA interaction by hexameric RNAs absent from the SARS-CoV-2 genomic RNA sequence. A. Structures of the N protein (NP), N-terminal RNA-binding domain (NTD) and NTD-deleted NP (NPΔNTD) are illustrated with the position of the domain. Intrinsically disordered regions (IDR) are shown. B. The probe RNA sequence (NC_045512.2, nt29733–29,764) is shown with the T7 promoter-driven nucleotide. Synthetic probe RNA was used for LLPS experiments. C. The inhibition of the NTD–RNA interaction by In-1, mIn-1, G6 and A6, determined by EMSA. The concentrations of the hexamers are indicated. Arrows indicate the positions of free probe RNA and RNA–NTD shifts. D. The IC50 of each hexamer was compared. The mean values and standard deviation (SD) of seven, six, four and three independent experiments are shown for In-1, mIn-1, G6 and A6, respectively. Variances were checked with F-tests, following which Student’s t-test was performed under the condition of unpaired equal variances (See Supplementary Fig. S1D). * and ** indicate P = 0.03 and P = 0.01, respectively.

Fig. 2. Phase separation of NP and NPΔNTD induced by the probe RNA and hexameric oligo-RNA. A. The 32-base RNA in the 3′ noncoding region of the SARS-CoV-2 genome was used as probe RNA. NP and NPΔNTD (25 and 50 μM) were mixed with 1.25 and 2.5 μM probe RNA or left without probe RNA (−) for 1.5 h and 17 h. DIC images were obtained. Scale bars of 10 μm are shown. Corresponding data for NTD are shown in Supplementary Fig. S2B. B. NP (25 μM) was mixed with 100 μM of hexameric oligo-RNA (A6, In-1 and mIn-1). C. G6 and G12 induced filamentous LLPS. NP (25 μM) was mixed with the indicated concentration of oligomeric RNA overnight.

Fig. 3. Effect of hexameric RNAs on the probe RNA–induced structures of NP and NPΔNTD. NP and NPΔNTD (25 μM each) were incubated with 2.5 μM of FITC-labeled 32-base probe RNA (FITC-probe RNA) for 3 h, followed by 50 μM of hexameric RNA (G6, A6, In-1 and mIn-1) for 16 h. DIC (DIC) and fluorescent (FL) images are shown. A scale bar of 10 μm is shown. Time- and concentration-dependent changes in the LLPS images (1/10 scale) and 25 μM and 50 μM hexameric RNA treatments, including the present data, are shown in Supplementary Fig. S3. Note that the fluidic phase changes of probe RNA–NPΔNTD (Fig. 2A) were weaker when FITC-probe RNA was used (compare Figs 2A and 3, 2.5 μM probe RNA versus 25 μM NPΔNTD).

Fig. 4. The FITC-probe RNA in the NP filamentous/droplet-like structures was excluded by guanosine 12-mer (G12). NP (25 μM) was mixed with 2.38 μM of FITC-labeled probe RNA for 45 min and further incubated with the indicated concentration of G12 oligomer for 75 min and 21 h. FITC-labeled and/or G12- controls are indicated as ‘-.’ DIC and fluorescent images were obtained at the indicated time. Time after the addition of probe RNA and, in parentheses, time after the addition of G12 are indicated. A scale bar of 10 μm is shown.

Fig. 5. The effect of the simultaneous addition of G12 and FITC-probe RNA on the phase separation of NP and NPΔNTD. A. G12 and FITC-probe RNA were mixed with NP simultaneously. B. Similar to A, FITC-probe RNA was mixed with NPΔNTD with G12 simultaneously (left) or 2 h later (right). Fluorescent images (FL) and DIC images (DIC) were obtained after 2, 5 and 18 h.

Fig. 6. The effects of phosphorothioate-modified guanosine 12-mer (G12(S)) and peptide nucleic acid guanosine hexamer (PNA-G6) on the phase separation of NP. A. NP (25 μM) was incubated with FITC-probe RNA (2.5 μM) for 2 h, then further incubated with the indicated concentration of the oligomers for 16 h. Corresponding data for NPΔNTD are shown in Supplementary Fig. S4. B. In-1 and G6 are not found in SARS-CoV-2 variants or most other coronavirus species. Variants’ names are indicated by Pango lineage. The GenBank accession numbers of the used sequences are shown. The numbers of sequences corresponding to G6 and In-1 appearing in the positive and negative strands of each virus are shown. An asterisk indicates one In-1 sequence found in the negative strand.