Systematic Evaluation of Bioorthogonal Reactions in Live Cells with Clickable HaloTag Ligands: Implications for Intracellular Imaging

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• 作者：Heather E. Murrey ; Joshua C. Judkins ; Christopher W. am Ende ; T. Eric Ballard ; Yinzhi Fang ; Keith Riccardi ; Li Di ; Edward R. Guilmette ; Joel W. Schwartz ; Joseph M. Fox ; Douglas S. Johnson
• 刊名：Journal of the American Chemical Society
• 出版年：2015
• 出版时间：September 9, 2015
• 年：2015
• 卷：137
• 期：35
• 页码：11461-11475
• 全文大小：935K
• ISSN：1520-5126

文摘

Bioorthogonal reactions, including the strain-promoted azide鈥揳lkyne cycloaddition (SPAAC) and inverse electron demand Diels鈥揂lder (iEDDA) reactions, have become increasingly popular for live-cell imaging applications. However, the stability and reactivity of reagents has never been systematically explored in the context of a living cell. Here we report a universal, organelle-targetable system based on HaloTag protein technology for directly comparing bioorthogonal reagent reactivity, specificity, and stability using clickable HaloTag ligands in various subcellular compartments. This system enabled a detailed comparison of the bioorthogonal reactions in live cells and informed the selection of optimal reagents and conditions for live-cell imaging studies. We found that the reaction of sTCO with monosubstituted tetrazines is the fastest reaction in cells; however, both reagents have stability issues. To address this, we introduced a new variant of sTCO, Ag-sTCO, which has much improved stability and can be used directly in cells for rapid bioorthogonal reactions with tetrazines. Utilization of Ag complexes of conformationally strained trans-cyclooctenes should greatly expand their usefulness especially when paired with less reactive, more stable tetrazines.