Selection and Characterization of SARS‐CoV‐2 Spike Binding Clickmers

Expanding the chemical repertoire of canonical nucleotides is key to unlocking the full functional potential of aptamers for diagnostic use. Herein, click-systematic evolution of ligands by exponential enrichment (SELEX) is employed to generate chemically modified DNA aptamers, termed clickmers, that target the SARS-CoV-2 spike (CoV2-S) glycoprotein. Two independent split-combine selection strategies yield distinct clickmer families functionalized with benzofuran or indole moieties. Lead candidates (BF1 and N2) demonstrate nanomolar affinity for wild-type CoV2-S and maintain binding to multiple variants, including Alpha, Delta, and Mu, as validated by flow cytometry, surface plasmon resonance, and microscale thermophoresis. Structure-function analysis reveals essential click-in positions for both full-length clickmers and a truncated N2 variant, as short as 31 nucleotides, which displays increased binding to the Omicron variant. These results highlight the versatility of the click-SELEX platform and exemplify its successful application to a clinically relevant target, advancing previous developments in the field.