Fig. 5: The functional importance of a long-range RNA-RNA interaction.

a Top, Arc plot showing a conserved long-range RNA-RNA interaction (LR1) that spans 17 kb in distance along the SARS-CoV-2 genome. Bottom, structure model of the LR1 interaction. The mutations for LR1A and LR1B mutant substitutions are indicated in green and orange, next to the original bases, respectively. Compensatory mutant LR1AB contains all the mutations for LR1A and LR1B to restore the proposed RNA structure. b Clustering analysis of the LR1 secondary structure ensemble reveals two populations of distinct structural morphology among the four clusters identified in the structural landscape (n = 1000). Long-range interaction structures in clusters C1 & C2 (dark and light blue) and clusters C3 & C4 (dark and light orange) are highly homologous, differing by several base pair variations. Structure conformations (C3 & C4) that maximize long-range base pairing are more numerous and more stable than conformations that maximize local base pairing (C1 & C2). c Bar-chart showing the viral load of SARS-CoV-2 WT and its mutants in the supernatants of infected Vero-E6 cells. Each column represents the mean ± SD of three technical replicates from one representative experiment. The same trends were observed in n = 3 independent experiments. P-values are calculated by two-sided t-test. d Diagram showing that antisense oligonucleotide (ASO_LR1) disrupts the original long-range base-pairing of LR1. e Workflow showing transfecting Vero-E6 cells with ASO, followed by SARS-CoV-2 WT or Omicron infection. Image was created in BioRender. Wang, J. (2025) https://BioRender.com/xy543s1. f, g Box-plot showing the amount of SARS-CoV-2 WT and Omicron RNAs in cells that were transfected with different ASOs, at indicated timepoints. The box represents the 25–75th percentiles, the median is the line inside. The whiskers show the minimum and maximum values. N = 2 independent experiments (total 6 replicates). ASO_CTRL: ASO that does not target any region on the viral genome, ASO_1: ASO that targets a randomly selected region (genome position 1563-1582), ASO_LR1: ASO that targets LR1 to disrupt the long-range base-pairing structure. P-values were calculated by two-sided Mann-Whitney test. Source data are provided as a Source Data file.