Targeting the Conserved RNA-Dependent RNA Polymerase with Antisense Oligonucleotides Suppresses SARS-CoV-2 Variants

The rapid evolution of Severe Acute Respiratory Syndrome Coronavirus 2(SARS-CoV-2) variants, particularly mutations in the spike protein, has compromised the efficacy of many therapeutics, highlighting the urgent need for broad-spectrum antiviral strategies. The RNA-dependent RNA polymerase (RdRp), a core enzyme essential for viral replication and transcription, represents an ideal target due to its high sequence conservation across SARS-CoV-2 variants and related coronaviruses, as well as its functional indispensability—mutations in its catalytic core typically incur significant viral fitness costs. In this study, we designed antisense oligonucleotides (ASOs) targeting the highly conserved RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2. Using RNAstructure v6.3 and OligoWalk for rational design based on secondary structure and binding thermodynamics, we identified several candidate ASOs. In plasmid-transfected cells, RDRP-5 potently knocked down RdRp mRNA expression. In a SARS-CoV-2 replicon system, it significantly suppressed viral replication. Furthermore, in live virus infection models, RDRP-5 effectively reduced viral RNA load and titers of multiple Variants of Concern, including the SARS-CoV-2 Wuhan-Hu-1, Delta, Omicron, and XBB.1.1.6. These findings establish RDRP-5 as a promising broad-spectrum ASO therapeutic candidate that targets an evolutionarily constrained region of RdRp, thereby overcoming variant-driven resistance. More broadly, this study validates the strategy of targeting conserved viral replication machinery as a potent approach to combat both current and future emerging coronaviruses, offering a potential pathway for rapid deployment against future pandemics.