Fig. 5: Single molecule characterization of mechanical properties of SARS-CoV-2 PRF RNA in the presence of ZAP-S.

A Schematic illustrating optical tweezers experiments. RNA was hybridized to single-stranded DNA handles flanking the SARS-CoV-2 frameshift site and conjugated to functionalized beads. A focused laser beam was used to exert pulling force from one end of the molecule. The force was gradually increased until the RNA was fully unfolded (bottom). B 3D structure of SARS-CoV-2 pseudoknot RNA (PK) derived from⁵¹ and colored according to the scheme used in Fig. 4. C Schematic representations of the RNAs studied. D–I Example unfolding and refolding traces of PK in the presence or absence of ZAP-S, “F” denotes the folded state, “I” the intermediate, and “U” the fully unfolded state, (D) PK (N = 273 FD curves from 24 molecules no ZAP-S, N = 219 FD curves from 24 molecules +ZAP-S samples), (E) ΔSL2 mutant (N = 146 FD curves from eight molecules no ZAP-S, N = 122 FD curves from eight molecules +ZAP-S samples), (F) ΔSL3 mutant (N = 127 FD curves from 12 molecules no ZAP-S, N = 163 FD curves from 11 molecules +ZAP-S samples), (G) ΔSL2 + 3 mutant (N = 216 FD curves from eight molecules no ZAP-S, N = 196 FD curves from 11 molecules +ZAP-S samples), (H) compensatory mutant (N = 158 FD curves from 12 molecules no ZAP-S, N = 169 FD curves from 16 molecules +ZAP-S samples), (I) PK in absence (blue) and presence (green) of IMP3 (N = 273 FD curves from 24 molecules no ZAP-S, N = 226 FD curves from 20 molecules +ZAP-S samples). J Distribution of refolding work in presence (pink) and absence (blue) of ZAP-S. K Normalized refolding work in the presence of ZAP-S or IMP3. Data points represent the mean ± s.d. (box) and min and max values (whiskers). P values were calculated using an ordinary unpaired one-sided ANOVA followed by Dunnett’s multiple comparisons test. * P < 0.05 – **** P < 0.00001. See also Supplementary Figs. 5, 6 and Supplementary Table 1.