Chemical Evolution of Double Covalent Aptamers for Sustained Protein Degradation and Improved Cytotoxicity in NK-Cell-Mediated Tumor Therapy.

Aptamers are single-stranded oligonucleotides that adopt intricate three-dimensional structures that accommodate high-affinity and high-specificity binding to a broad range of targets, typically through noncovalent bonding, such as π-π stacking and electrostatic interactions. However, covalent small-molecule drugs that bind to specific proteins can increase biological efficiency, potency, and duration compared with conventional noncovalent interactions. Nonetheless, the biological effects of covalent target binding by aptamers remain largely unexplored owing to the challenge of designing aptamers capable of efficient covalent interactions. Herein, we addressed this challenge by leveraging the capability of structure prediction enabled by AlphaFold3 and molecular docking alongside mutation-and-activity validations to chemically engineer sgc8c, a DNA aptamer targeting protein tyrosine kinase 7 (PTK7). Through introducing dual covalent warheads, including the incorporation of three nucleobase modified deoxyuridines bearing UV-inducible diazirine groups and the installation of arylsulfonyl fluoride (SF) warheads on three phosphorothioate (PS) internucleotide linkages, we successfully configured a dual-covalent sgc8c mutant, denoted DC-sgc8c, capable of specific PTK7 binding and proximity-enabled cross-linking in buffer and on cancer cell surfaces. DC-sgc8c exhibited significantly increased biostability, promoted efficient and sustained PTK7 degradation via a lysosome-targeting chimera (LYTAC), and potentiated NK cell cytotoxicity upon membrane anchoring. The generalizability and versatility of predicted aptamer-protein structure directed covalent aptamer design strategy was further proven by tuning the nucleobase attached diazirine warhead of sgc8c to SF installed to backbone PS and by attaining covalent SL1 variants bearing backbone derivatized SF that can cross-link with its target c-Met. Our approach provides a proof-of-principle platform for converting conventional noncovalent aptamers into covalent counterparts through model-guided placement of reactive warheads, supplemented by systematic experimental validation, thereby achieving irreversible target intervention with therapeutic potential beyond the reach of noncovalent aptamers.