Unwinding the SARS-CoV-2 Ribosomal Frameshifting Pseudoknot with LNA and G-Clamp-Modified Phosphorothioate Oligonucleotides Inhibits Viral Replication

Ribosomal frameshifting (RFS) at the slippery site of SARS-CoV-2 RNA is essential for the biosynthesis of the viral replication machinery. It requires the formation of a pseudoknot (PK) structure near the slippery site and can be inhibited by PK-disrupting oligonucleotide-based antivirals. We obtained and compared three types of such antiviral candidates, namely locked nucleic acids (LNA), LNA-DNA gapmers, and G-clamp-containing phosphorothioates (CPSs) complementary to PK stems. Using optical and electrophoretic methods, we showed that stem 2-targeting oligonucleotide analogs induced PK unfolding at nanomolar concentrations, and this effect was particularly pronounced in the case of LNA. For the leading PK-unfolding LNA and CPS oligonucleotide analogs, we also demonstrated dose-dependent RSF inhibition in dual luciferase assays (DLAs). Finally, we showed that the leading oligonucleotide analogs reduced SARS-CoV-2 replication at subtoxic concentrations in the nanomolar range in two human cell lines. Our findings highlight the promise of PK targeting, illustrate the advantages and limitations of various types of DNA modifications and may promote the future development of oligonucleotide-based antivirals.

Automated summary

Ribosomal frameshifting at the slippery site of SARS-CoV-2 RNA is crucial for viral replication, and inhibition of this process by oligonucleotide-based antivirals shows promise. In this study, locked nucleic acids (LNA) and other oligonucleotide analogs targeting the pseudoknot near the slippery site were shown to induce PK unfolding and inhibit frameshifting in a dose-dependent manner. These analogs also demonstrated the ability to reduce SARS-CoV-2 replication at subtoxic concentrations in human cell lines, highlighting their potential as antiviral agents.