Sofosbuvir terminated RNA is more resistant to SARS-CoV-2 proofreader than RNA terminated by Remdesivir

Figure 1. Comparison of structures of the prodrugs (a) Sofosbuvir (SOF) and (b) Remdesivir (RDV) and their active triphosphate forms (SOF-TP and RDV-TP). Top: Prodrug (phosphoramidate) forms; Bottom: Active triphosphate forms.

Figure 2. Polymerase reactions with SOF-TP, UTP or UTP + RDV-TP to produce RNA extension products. The sequence of the RNA template-loop-primer used for these polymerase extension reactions is shown at the top of the figure. (a) MALDI-TOF mass spectrum of the unextended RNA. Polymerase extension reactions were performed by incubating (b) SOF-TP, (c) UTP and (d) UTP + RDV-TP with pre-assembled SARS-CoV-2 polymerase (nsp12, nsp7 and nsp8) and the indicated RNA template-loop-primer, followed by detection of reaction products by MALDI-TOF MS. The accuracy for m/z determination is approximately ± 10 Da.

Figure 3. Treatment of the RNA products with exonuclease and analysis by MALDI-TOF MS to determine relative excision of Sofosbuvir, UMP and Remdesivir. Untreated products (0 min) are shown in (a) for SOF extended RNA, (d) for UMP extended RNA and (g) for UMP plus RDV extended RNA. Exonuclease reactions were performed by incubating the purified RNA products, generated using the same procedure as in Fig. 2, with pre-assembled SARS-CoV-2 exonuclease complex (nsp14 and nsp10) for 5 min (b,e,h) or 30 min (c,f,i), followed by detection of reaction products by MALDI-TOF MS. The signal intensities were normalized to the highest peak within each time series. The accuracy for m/z determination is approximately ± 10 Da.

Figure 4. Treatment of the RNA products with exonuclease and then combined for analysis by MALDI-TOF MS to determine relative excision of Sofosbuvir, UMP and Remdesivir. Untreated products (0 min) are shown in (a) for SOF and UMP extended RNA and (c) for SOF and UMP + RDV extended RNA. Exonuclease reactions were performed by incubating the purified RNA products with pre-assembled SARS-CoV-2 exonuclease complex (nsp14 and nsp10). The SOF and UMP extended RNAs were treated with the exonuclease complex for 15 min and then combined for purification followed by MALDI-TOF–MS (b). The insets in (a,b) are an enlargement of the 8200–8300 Da portion of the spectrum. The SOF and UMP + RDV extended RNAs were treated with the exonuclease complex for 5 min or 30 min and combined for purification and MALDI-TOF–MS (d,e, respectively). The signal intensities were normalized to the highest peak within each time series. The accuracy for m/z determination is approximately ± 10 Da.