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.