DNA oligonucleotides block viral entry of SARS-CoV-2 Omicron variants

Successful viruses including SARS-CoV-2 antagonize and evade immune detection to establish infection. As population immunity to the ancestral strain has risen, new variants-particularly Omicron-have evolved to avoid detection by both circulating antibodies and the innate immune system. Screening innate immune agonists, we previously identified STING agonists as potent inhibitors of the ancestral variant of SARS-CoV-2. When we explored the sensitivity of Omicron BA.1 to diverse innate stimuli, we found that Omicron is also sensitive to interferons and STING agonists; however, Omicron variants are uniquely restricted by small DNA oligonucleotide ligands known to activate TLR9. Mechanistic studies showed that short, but not long, DNA oligonucleotides of diverse sequences, including those that do not engage TLR9, block Omicron infection at the entry step downstream of TMPRSS2-mediated activation. Using genetic and pharmacological assays we confirmed that activity was independent of TLR9. Furthermore, these oligodeoxynucleotides (ODNs) are active in vivo, reducing SARS-CoV-2 Omicron titers. Together, these findings identify a previously unrecognized antiviral activity of short DNA oligonucleotides and establish them as selective inhibitors of SARS-CoV-2 Omicron entry.