Genetically Targetable and Color‐Switching Fluorescent Probe

Zhang, Ming; Yan, Qi; Waggoner, Alan S.; Bruchez, Marcel P.

doi:10.1002/cbic.201200334

Cited by 18 publications

(30 citation statements)

References 69 publications

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“…Acid catalyzed deprotection of the Boc group with 1N HCl yielded the mono-sulfonated intermediate MG[H]-EDA-Lys-Tau 10 , a valuable compound for the preparation of tandem dyes. 19, 20 MG[H]-EDA-Lys-Tau 10 was again coupled with tetrabutylammonium 2-({4-[(2,5-dioxopyrrolidin-1-yl)oxy]-4-oxobutanoyl}amino) ethane sulfonate 8 under anhydrous conditions, and without isolation oxidized with tetrachlorobenzoquinone to give the target compound MG-B-Tau 1 . This compound is very water-soluble and thus was purified by reversed phase chromatography.…”

Section: Resultsmentioning

confidence: 99%

Near-instant surface-selective fluorogenic protein quantification using sulfonated triarylmethane dyes and fluorogen activating proteins

et al. 2015

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Summary Agonist-promoted G-protein coupled receptor (GPCR) endocytosis and recycling plays an important role in many signaling events in the cell. However, the approaches that allow fast and quantitative analysis of such processes still remain limited. Here we report an improved labeling approach based on the genetic fusion of a Fluorogen Activating Protein (FAP) to a GPCR and binding of a sulfonated analog of the malachite green (MG) fluorogen to rapidly and selectively label cell surface receptors. Fluorescence microscopy and flow cytometry demonstrate that this dye does not cross the plasma membrane, binds with high affinity to a dL5** FAP-GPCR fusion construct, activating tagged surface receptors within seconds of addition. The ability to rapidly and selectively label cell surface receptors with a fluorogenic genetically encoded tag allows quantitative imaging and analysis of highly dynamic processes like receptor endocytosis and recycling.

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Section: Resultsmentioning

confidence: 99%

Near-instant surface-selective fluorogenic protein quantification using sulfonated triarylmethane dyes and fluorogen activating proteins

et al. 2015

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“…In a second example, linking MG to tetramethylrhodamine yields D-A F bichromophores with efficient FRET and red emission. 26 The authors also reported an interesting feature: the equilibrium between nonfluorescent carbinol and fluorogenic triphenylmethane forms of MG depends strongly on the nature of the linkage between the two chromophores, allowing tuning of the emission between yellow-orange (TMR) and red (MG).…”

Section: Discussionmentioning

confidence: 99%

Bichromophoric dyes for wavelength shifting of dye-protein fluoromodules

et al. 2015

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Dye-protein fluoromodules consist of fluorogenic dyes and single chain antibody fragments that form brightly fluorescent noncovalent complexes. This report describes two new bichromophoric dyes that extend the range of wavelengths of excitation or emission of existing fluoromodules. In one case, a fluorogenic thiazole orange (TO) was attached to an energy acceptor dye, Cy5. Upon binding to a protein that recognizes TO, red emission due to efficient energy transfer from TO to Cy5 replaces the green emission observed for monochromophoric TO bound to the same protein. Separately, TO was attached to a coumarin that serves as an energy donor. The same green emission is observed for coumarin-TO and TO bound to a protein, but efficient energy transfer allows violet excitation of coumarin-TO, versus longer wavelength, blue excitation of monochromophoric TO. Both bichromophores exhibit low nanomolar KD values for their respective proteins, >95% energy transfer efficiency and high fluorescence quantum yields.

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“…Some breakthroughs occurred in the covalent bio-conjugate field, where the same target ligand for capture may be chemically coupled with unique color fluorophores, a very similar approach to using commercially labeled antibodies for labeling cells (Chen et al, 2007;Kosaka et al, 2009;Vivero-Pol et al, 2005;Lee et al, 2010;Liu et al, 2014;Uttamapinant et al, 2010;Wombacher et al, 2010;Yao et al, 2012). Likewise, other groups have utilized bio-conjugate platforms based on tandem dye interactions that have resulted in fluorescence resonance energy transfer (FRET), a donor-acceptor approach that amplifies the Stokes shift of a molecule resulting in fluorescence emissions at longer wavelengths (Brun et al, 2009(Brun et al, , 2011Gallo et al, 2015;Pham et al, 2015;Rajapakse et al, 2010;Robers et al, 2009;Saunders et al, 2014;Yushchenko et al, 2012;Zürn et al, 2010). As a result, we find that current methods prove lacking in multi-color detection and real-time signal modulation.…”

Section: Introductionmentioning

confidence: 99%

Breaking the color barrier – a multi-selective antibody reporter offers innovative strategies of fluorescence detection

Gallo

Jarvik

2017

Journal of Cell Science

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A novel bi-partite fluorescence platform exploits the high affinity and selectivity of antibody scaffolds to capture and activate small-molecule fluorogens. In this report, we investigated the property of multiselectivity activation by a single antibody against diverse cyanine family fluorogens. Our fluorescence screen identified three cellimpermeant fluorogens, each with unique emission spectra (blue, green and red) and nanomolar affinities. Most importantly, as a protein fusion tag to G-protein-coupled receptors, the antibody biosensor retained full activity -displaying bright fluorogen signals with minimal background on live cells. Because fluorogen-activating antibodies interact with their target ligands via non-covalent interactions, we were able to perform advanced multi-color detection strategies on live cells, previously difficult or impossible with conventional reporters. We found that by fine-tuning the concentrations of the different color fluorogen molecules in solution, a user may interchange the fluorescence signal (onset versus offset), execute real-time signal exchange via fluorogen competition, measure multi-channel fluorescence via colabeling, and assess real-time cell surface receptor traffic via pulsechase experiments. Thus, here we inform of an innovative reporter technology based on tri-color signal that allows user-defined fluorescence tuning in live-cell applications.

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Genetically Targetable and Color‐Switching Fluorescent Probe

Cited by 18 publications

References 69 publications

Near-instant surface-selective fluorogenic protein quantification using sulfonated triarylmethane dyes and fluorogen activating proteins

Near-instant surface-selective fluorogenic protein quantification using sulfonated triarylmethane dyes and fluorogen activating proteins

Bichromophoric dyes for wavelength shifting of dye-protein fluoromodules

Breaking the color barrier – a multi-selective antibody reporter offers innovative strategies of fluorescence detection

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