Abstract
The SARS-CoV-2 spike (S) protein, crucial for viral entry, remains a key target for diagnostics and therapeutics amid evolving variants. Here, we describe the selection and characterization of novel DNA aptamers targeting the S1 subunit, including the Omicron strain, via systematic evolution of ligands by exponential enrichment (SELEX) and biolayer interferometry (BLI). Three aptamers-AptS1-tSP4, AptS1-tSP10, and AptS1-tSP11-exhibited nanomolar dissociation constants (14-59 nM), with AptS1-tSP10 demonstrating good selectivity over MERS-CoV and robust binding in human saliva and pseudovirus samples. Integration with proximity ligation assay and qPCR (PLA-qPCR) achieved a detection limit of 3 pM, surpassing many antibody-based methods. Mass spectrometry-based epitope mapping identified the receptor-binding domain (RBD) peptide VGGNYNYLYR as the primary binding site for AptS1-tSP10. Molecular dynamics and quantum mechanics simulations revealed stable interactions through hydrogen bonding and π-π stacking with neutral residues in both open and closed spike conformations, independent of variant mutations. These multifunctional aptamers offer a versatile platform for ultrasensitive, epitope-specific SARS-CoV-2 detection and pave the way for nucleic acid-based therapeutics to combat viral infections.