Fig. 4. In vitro characterization of ZAP-S interaction with SARS-CoV-2 –1 PRF RNA.
A Proposed structure of the PRF element of SARS-CoV-2. Nucleotide substitutions in the compensatory mutant are indicated (arrowheads). B Schematic representations of the RNAs studied. C–H Microscale thermophoresis assay to monitor ZAP-S binding to (C) Full PRF, (D) ΔSL2 mutant, (E) ΔSL3 mutant, (F) ΔSL2 + 3 mutant, (G) compensatory mutant, (H) scrambled mutant. Unlabeled protein (40 pM–2 µM) was titrated against 3′ pCp-Cy5 labeled RNA (5 nM) and thermophoresis was recorded at 25 °C with 5% LED intensity and medium MST power. Change in fluorescence (ΔFnorm) was measured at MST on-time of 2.5 s. Data were analyzed for ΔFnorm using standard functions of MO. Affinity Analysis software and data was plotted and KD was determined using Graphpad Prism 9.2.0. Data represent mean ± s.d. of three measurements (n = 3). For the related thermophoretic traces, see also Supplementary Fig. 4A-F. For the related DNA sequences of the mutants, see also Supplementary Table 2.