铜催化点击反应:DNA增强配体可实现与铜催化剂的活细胞内点击化学反应。

inCu-click: DNA-enhanced ligand enables live-cell, intracellular click chemistry reaction with copper catalyst.

作者信息

Nian Keqing, Liu Yifang, Qiu Yuchen, Zhang Zhuoyu, Brigandi Laura, Wanunu Meni, Rouhanifard Sara H

机构信息

Dept. of Bioengineering, Northeastern University, Boston, MA, USA.

Dept. of Physics, Northeastern University, Boston, MA, USA.

出版信息

Nat Commun. 2025 May 23;16(1):4788. doi: 10.1038/s41467-025-60143-3.

Abstract

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.

摘要

通过铜催化的叠氮化物-炔烃环加成反应(CuAAC)标记细胞生物分子具有快速的反应动力学,并且使用的小叠氮化物和炔烃探针能最小程度地干扰分子功能,这使其成为追踪生物分子的理想选择。然而,在活细胞内应用CuAAC受到所需高铜水平的阻碍,这会损害细胞健康。为了克服这一障碍,在此我们开发了inCu-click,一种细胞内CuAAC方法,该方法采用与DNA偶联的配体(BTT-DNA)将铜离子定位并浓缩在反应位点。这种设计允许在低细胞内铜浓度下进行高效的点击化学反应,无需添加铜盐,并支持模板驱动的配体接近以及脂质体将配体递送至细胞内。在此我们表明,inCu-click能够对活细胞中新生的磷脂和蛋白质进行稳健的荧光标记,对细胞活力的影响可忽略不计,从而建立了一个在复杂的活细胞环境中实时可视化生物分子动态的平台。

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