Figure 1. Visualizing SARS-CoV-2 RNA with CoronaFISH.

(A) Principle of CoronaFISH. 96 primary probes are prehybridized in vitro with dye-carrying secondary probes via the shared FLAP sequence. Resulting duplexes are subsequently hybridized in cells to target the SARS-CoV-2 positive or negative RNA. (B) Replication cycle of SARS-CoV-2. Incoming, genomic positive-strand RNA (green) is used to produce viral polymerase. Polymerase produces a negative-strand replication intermediate (violet), which serves as a template for synthesis of full-length positive-strand and shorter subgenomic RNAs (green). The latter are used to produce other viral proteins. (C) Genome of SARS-CoV-2 with indicated probe positions targeting the positive and negative strand. (D) CoronaFISH images of uninfected and infected Vero cells with either the positive (top) or negative (bottom) strands detected with Cy3-labeled probes (white). Nuclei are labeled with DAPI (blue). Shown are zoom-ins on individual cells. Full-size images are shown in Fig S1A. First column shows uninfected control cells, second and third columns show infected cells with different intensity scaling as indicated in brackets (the first and second values in brackets indicate the pixel values corresponding to the lowest and brightest intensities in the displayed image, respectively). Scale bars: 5 μm. Scale bar in red inset: 1 μm. (E) Quantification of signal intensities in individual cells. Dashed line is the 99% quantile estimated from uninfected samples. (F) CoronaFISH image of the positive strand combined with immunofluorescence detection of dsRNA with the J2 antibody. Scale bar in full image: 2 μm, in inset: 0.5 μm. (G) Dual-color imaging of positive and negative strands. Scale bar in full image: 10 μm, in inset: 2 μm. Intensity scalings in panels (F) and (G) are shown as described for panel (D).