Bichromophoric dyes for wavelength shifting of dye-protein fluoromodules

Abstract

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.

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

“…As a result, since the scFv HL1.0.1-TO1 was previously isolated against a monomeric fluorogen, we suggest that only one tandem-half of the molecule may actually form the affinity complex rather than the whole-molecule tandem fluorogen. A similar observation was previously determined when using tandem pairings of fluorogen-fluorophore or fluorogenpolyethylene-biotin molecules (Pham et al, 2015;Szent-Gyorgyi et al, 2010;Vasilev et al, 2016). In these three studies, the fluorogen component shows affinity interaction with the scFv irrespective of its tandem fluorogen pair, where most likely both moieties display independent functional activities.…”

“…Here, the antibody functions as a protein cage that sterically confines the smallmolecule fluorogen, and, upon light excitation, the fluorogen emits fluorescence due to non-radiative energy decay and energy release. Incidentally, FAP technology also offers a malleable approach for altering fluorescence signals, primarily by modifying the chemical composition of the synthetic fluorogens in order to tune their binding affinities and/or spectra (Pham et al, 2015;Rastede et al, 2015;Saunders et al, 2013Saunders et al, , 2014Szent-Gyorgyi et al, 2010). Furthermore, FAP reporters have demonstrated a rapid advancement as tools for labeling targets at the surface of cells (Fig.…”

“…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.…”

Dark dyes–bright complexes: fluorogenic protein labeling

“…This fluorogen could be linked to a variety of dye acceptors[36], including pH sensors and far-red energy transfer acceptors, producing multicolor labeling reagents. Grover et al prepared a pH senstitive analog of the TO1 dye, which reported in real-time on changes in GPCR endocytosis and intracellular acidification of the endosomes[37].…”

Design, synthesis and application in biological imaging of a novel red fluorescent dye based on a rhodanine derivative

“…Rhodanine, [23][24][25] a ve-membered S,N-heterocycle, can serve as a building block for organic dyes in biomedical elds such as biomolecule uorescent labeling, 11 proteomics, 12 photodynamic therapy, [13][14][15] live cell imaging, [16][17][18][19] antitumor drugs, 20 DNA detection, 8 pH probes, 21 uorescent sensors, 22 etc. Owning to their strong electron-withdrawing properties, rhodanine derivatives have been broadly utilized as acceptors for the design of donor-acceptor (D-A) type photovoltaic materials in organic solar cells (OSCs).…”