Labeling cellular biomolecules via copper-catalyzed azide-alkyne cycloaddition (CuAAC) offers rapid reaction kinetics and uses small azide and alkyne probes that minimally disturb molecular function, making it ideal for tracking biomolecules. However, applying CuAAC inside living cells has been hindered by the high copper levels required, which compromise cell health. To overcome this barrier, here, we develop inCu-click, an intracellular CuAAC approach that employs a DNA-conjugated ligand (BTT-DNA) to localize and concentrate copper ions at the reaction site. This design permits efficient click chemistry at low intracellular copper concentrations without added copper salts and supports template-driven proximity and liposomal delivery of the ligand into cells. Here we show that inCu-click enables robust fluorescent labeling of nascent phospholipids and proteins in live cells with negligible impact on viability, establishing a platform for real-time visualization of biomolecule dynamics in complex, live cell environments.

© 2025. The Author(s).

通过铜催化的叠氮化物-炔烃环加成（CuAAC）对细胞生物分子进行标记，提供了快速的反应动力学，并使用了最小程度干扰分子功能的小型叠氮化合物和炔类探针，使其非常适合用于追踪生物分子。然而，在活细胞内应用CuAAC受到了所需高铜水平的阻碍，这会影响细胞健康。为了克服这一障碍，我们开发了一种名为inCu-click的细胞内CuAAC方法，该方法采用DNA连接配体（BTT-DNA）将铜离子定位并集中于反应位点。这种设计允许在没有添加铜盐的情况下，在低细胞内铜浓度下高效地进行点击化学，并支持模板驱动的邻近和脂质体递送配体进入细胞。在这里，我们展示了inCu-click能够使新生磷脂和蛋白质在活细胞中实现稳健的荧光标记，对存活率几乎没有影响，从而建立了一个平台，在复杂的活细胞环境中实时可视化生物分子动态。

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