期刊
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
卷 137, 期 35, 页码 11461-11475出版社
AMER CHEMICAL SOC
DOI: 10.1021/jacs.5b06847
关键词
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资金
- NIH from NIBIB [R01EB014354]
- NIH [P20GM104316, P30GM110758, S10RR026962-01, S10OD016267-01]
- NSF [CHE 0840401, CHE-1229234]
- Division Of Chemistry
- Direct For Mathematical & Physical Scien [1229234] Funding Source: National Science Foundation
Bioorthogonal reactions, including the strain-promoted azide-alkyne cycloaddition (SP.AAC) and inverse electron demand Diels-Alder (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 bio-orthogonal 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.
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