In the assays, the RdRp catalytic domain of RNA-directed RNA polymerase (Reference: PX-COV-P006, ProteoGenix, Schiltigheim, France) was used. As a template, we used the Genomic RNA from 2019 Novel Coronavirus, Strain: 2019-nCoV/USA-WA1/2020 (Reference: ATCC-VR-1986D, ATCC, Wessel, Germany), while primers (RNA or DNA) complementary to specific regions were obtained from Eurofins. In the reactions, we used the 10X First-Strand Buffer and RNase Inhibitor from the Amino Allyl MessageAmp™ II aRNA Amplification Kit (Reference: AM1753, Thermo Fisher Scientific, Bremen, Germany), as well as dNTPs/rNTPs. A commercial inhibitor Cordycepin 5′-triphosphate sodium salt (Reference: C9137, Sigma-Aldrich, Taufkirchen, Germany) was used as a positive control. The run conditions were 37 °C for 2 h, and samples were run on agarose gels.

The positive sample included everything mentioned above (without the inhibitor), as well as commercial nucleotides, to verify that the synthesis is performed without any problems, while a second positive control, including the commercial inhibitor, was used to compare our analogues and their ability to terminate the reaction. Negative controls, including only primer, or without specific nucleotides, were used as well. In the reactions, where our nucleoside analogues were added, commercial normal nucleotides were also added at a ratio of 1:1. The products were run on agarose gels using an appropriate ladder. Gels were stained with MidoriGreen Dye, which stains dsDNA, ssDNA, dsRNA and ssRNA.

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