Development of RNA Aptamers that Inhibit the RNA-dependent RNA Polymerase Activity of SARS-CoV-2 Strains

The continuous emergence of SARS-CoV-2 variants with enhanced transmissibility and immune escape capability highlights the urgent need for mutation-independent anti-viral strategies. SARS-CoV-2 non-structural protein 12 (NSP12), encoding RNA-dependent RNA polymerase (RdRp), plays a pivotal role in viral replication and is a highly con-served therapeutic target. In this study, we developed RNA aptamers targeting NSP12 us-ing the SELEX (Systematic Evolution of Ligands by EXponential enrichment) approach. SELEX was performed with purified NSP12 protein from the Omicron variant and led to the identification of aptamer candidates with high binding ability. RNA–protein pull-down assays confirmed binding between representative aptamers and NSP12 with high affinity. Competition assays supported specific binding between aptamers and NSP12. Of note, functional validation using a primer extension assay revealed that the aptamers effectively inhibited NSP12 RdRp activity. Furthermore, the aptamers consist-ently bound to and inhibited NSP12 variants from wild-type, Alpha, Delta, and Omicron strains. These results suggest that the selected RNA aptamers act as broad-spectrum in-hibitors that target a conserved region of NSP12 and offer a promising platform for the development of anti-viral agents against current and future SARS-CoV-2 variants and other RNA viruses.