Aptamer-Directed Dual-Site Photoaffinity Labeling Enables Functional Regulation of Protein–Protein Interactions

Direct, site-specific modification of native membrane proteins without genetic manipulation remains a significant challenge.Here, we introduce an Aptamer-directed Dual-site Photoaffinity Labeling (ADPAL) strategy that enables precise and functional modification of membrane proteins in their native context.This approach integrates aptamer-based recognition with proximity-activated photoreactive chemistry to achieve dual-site covalent labeling with high specificity and efficiency.As a proof of concept, we applied the ADPAL strategy to both isolated proteins, lysozyme and thrombin, as well as to membrane proteins on living cells, including the mesenchymal-epithelial transition factor (Met) and protein tyrosine kinase 7 (PTK7) receptors.These dual-site modifications triggered protein self-assembly, reprogrammed protein-protein interactions, facilitated protein cluster formation, and modulated cellular functions.Compared to conventional noncovalent or single-site covalent labeling methods, ADPAL offers enhanced specificity and regulatory control.This work establishes ADPAL as a versatile and programmable platform for precise membrane protein labeling and functional manipulation, with broad potential for biological research and therapeutic development.