Primer Extension-Mediated Aptamer-Gated DNA-Encoded Library Screening for Physiologically Relevant Ligand Discovery

DNA-encoded library (DEL) technologies have revolutionized drug discovery, yet their reliance on purified targets often yields ligands with a poor physiological relevance. Here, we introduce Primer Extension-Mediated Aptamer-Gated DEL Screening (PEMAG-DEL), a transformative platform that integrates aptamer-guided target localization, enzymatic fidelity filtering, and combinatorial chemistry to enable ligand discovery in native-like environments. We selected carbonic anhydrase II (CA II) as a model target and designed a 125-member DEL library composed of peptide–sulfonamide conjugates to perform screening and selection using the PEMAG-DEL platform. As a result, we identified a high-affinity ligand, Sulfo–His–His–Ala, which exhibited a 16.3-fold improvement in binding affinity (IC50 = 0.84 μM) compared to the parent compound, 4-sulfobenzoic acid. PEMAG-DEL screening in HeLa cell lysates demonstrated robust resistance to nonspecific interference from membrane proteins and aggregates. These findings confirm that PEMAG-DEL enables precise selection of biologically relevant ligands even in complex, native-like environments.