Open AccessSystematic Evaluation of Bioorthogonal Reactions in Live Cells with Clickable HaloTag Ligands: Implications for Intracellular Imaging
Author(s) -
Heather E. Murrey,
Joshua C. Judkins,
Christopher W. am Ende,
T. Eric Ballard,
Yinzhi Fang,
Keith Riccardi,
Li Di,
Edward Guilmette,
Janet Schwartz,
Joseph M. Fox,
Douglas S. Johnson
Publication year - 2015
Publication title -
journal of the american chemical society
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.115
H-Index - 612
eISSN - 1520-5126
pISSN - 0002-7863
DOI - 10.1021/jacs.5b06847
Subject(s) - bioorthogonal chemistry , chemistry , reagent , tetrazine , context (archaeology) , cycloaddition , reactivity (psychology) , click chemistry , combinatorial chemistry , live cell imaging , cell , biochemistry , organic chemistry , catalysis , medicine , paleontology , alternative medicine , pathology , biology
Bioorthogonal reactions, including the strain-promoted azide-alkyne cycloaddition (SPAAC) 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 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.