inCu-click: Enabling copper-catalyzed click chemistry inside living cells

Among the many strategies for bioconjugation, Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) is a particularly powerful click chemistry tool due to its rapid kinetics and the commercial availability of a wide range of conjugates. Although CuAAC has been widely adopted for labeling biomolecules on living cell surfaces, intracellular labeling in live cells remains challenging. The elevated copper concentration needed to drive the CuAAC reaction efficiently can compromise cell death, restricting intracellular CuAAC labeling to cross-linked cell systems. To address this limitation, inCu-click, an intracellular CuAAC platform that utilizes a DNA-conjugated ligand (inCu-click ligand) to localize and concentrate copper ions at the intended reaction site, was developed. The inCu-click system enables efficient click chemistry under reduced intracellular copper levels, without the need for additional copper salts. This design supports liposomal delivery of the ligand into cells for intracellular CuAAC labeling. Using this platform, we demonstrate reliable and robust fluorescent labeling of nascent phospholipids and alkyne drugs in live cells with minimal impact on viability. Collectively, inCu-click provides a practical, broadly applicable platform for real-time visualization of biomolecular dynamics in complex live-cell environments.