DNA Oligonucleotides Block Viral Entry of SARS-CoV-2 Omicron Variants
Jorge A. Acuña, Benjamin Gabriel, Kasirajan Ayyanathan, Jesse Miller, Yue Li, Kanupriya Whig, Yongqing Zhu, Marisa McGrath, Peter Hewins, Kellie Ann Jurado, Matthew B. Frieman, David C. Schultz, Sara Cherry
Abstract
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.
Introduction
SARS-CoV-2 continues to evolve under immune pressure, giving rise to variants with enhanced transmissibility and immune evasion. Mutations in the Spike protein have been central to this process, enabling variants such as Alpha (B.1.1.7), Delta (B.1.617.2), and especially Omicron (ex. BA.1) to escape antibodies and impact cell-entry mechanisms. All SARS-CoV-2 variants bind to the ACE2 receptor and require subsequent Spike cleavage by either TMPRSS2 at the plasma membrane or cathepsins in endosomes, to trigger fusion between the viral and cellular membranes, releasing viral RNA into the cytoplasm.
Materials and Methods:
Calu-3 cells (American Type Culture Collection, ATCC, HTB-55) were cultured in minimum essential medium supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) non-essential amino acids, 1% (v/v) penicillin/streptomycin, and 1% (v/v) Gltuamax (Invitrogen) at 37°C, and 5% CO2. Caco-2 cells (ATCC, HTB-37) were cultured in MEM alpha supplemented with 20% (v/v) fetal bovine serum, 1% (v/v) penicillin–streptomycin and 1% (v/v) l-glutamine. A549-ACE2 cells were cultured in RPMI-1640, 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1% Glutamax.
Discussion:
We compared the activity of a broad panel of innate immune agonists against the ancestral SARS-CoV-2 WA1 strain and the Omicron BA.1 variant to determine whether these lineages differ in their sensitivity to innate immune activation. Although both viruses responded similarly to interferons, STING agonists, and polyI:C, we identified the TLR9 agonist ODN2006—a synthetic short single-stranded oligodeoxynucleotide—as a potent inhibitor of Omicron BA.1 but not WA1. This striking lineage specificity prompted us to define the mechanism underlying the selective inhibition of Omicron by ODN2006.
Acknowledgments:
We acknowledge members of the Cherry lab for input and discussion. We thank the members of the University of Pennsylvania High Throughput Screening Core (RRID: SCR_022379) for technical support. We thank members of the Jurado lab for input and discussion regarding mice experiments.
Citation: Acuña JA, Gabriel B, Ayyanathan K, Miller J, Li Y, Whig K, et al. (2026) DNA oligonucleotides block viral entry of SARS-CoV-2 Omicron variants. PLoS Pathog 22(7): e1014461. https://doi.org/10.1371/journal.ppat.1014461
Editor: Roberto Cattaneo, Mayo Clinic, UNITED STATES OF AMERICA
Received: January 22, 2026; Accepted: July 9, 2026; Published: July 30, 2026.
Copyright: © 2026 Acuña et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All data is available in the manuscript.
Funding: This work was supported by grants from the National Institutes of Health to S.C. (1-R01-AI-150246, 1-R01-AI-152362, 1-R01-AI-140539). S.C. was a recipient of the Burroughs Wellcome Investigators in the Pathogenesis of Infectious Disease Award and was supported by the Deans Innovation Fund as well as the Linda and Laddie Montague Foundation. J.A.A. is supported by 5F31AI183630-02 from the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
