Aptamer-Guided, Hydrolysis-Resistant Deoxyoxanosine Enables Epitope- and Moiety-Selective Conjugation to Nonengineered Proteins Even in Complex Environments

초록

In protein engineering, researchers have extensively explored the incorporation of nonprotein entities into proteins to extend their functionalities to various applications; however, achieving precise modifications of proteins is still challenging. This study demonstrates epitope- and moiety-selective conjugation of nonengineered proteins by integrating "slow-reactive and hydrolysis-resistant" deoxyoxanosine (dOxa) into a "target- and epitope-selective" aptamer. The amine-reactive dOxa-containing aptamers are dominantly single-lysine-selective at recognition sites, achieving significantly high conjugation yields with remarkably low off-target reactions in complex environments under near-physiological conditions through a catalyst-free, one-pot reaction. When stoichiometrically controlled protein-DNA conjugates are efficiently produced for various proteins, high conjugation selectivity enables semipermanent regulation of enzymatic functions, targeted labeling in a protein mixture, and even heterofunctionalization of a single protein. As our dOxa-containing aptamers selectively react with the recognition sites of target proteins among nontargets, we demonstrate bioorthogonal labeling of live-cell surface nucleolin and PTK7 in amine-rich cell media, displaying their distinct distributions. Aptamer-guided dOxa positioning offers a promising strategy for site-specific modification of native proteins in complex environments, opening new avenues for the synergistic collaboration between nucleic acids and proteins.

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