Fluorogenic dendrons with multiple donor chromophores as bright genetically targeted and activated probes.
We have developed a class of dendron-based fluorogenic dyes (termed dyedrons) comprised of multiple cyanine (Cy3) donors coupled to a single malachite green (MG) acceptor that fluoresce only when the MG is noncovalently but specifically bound to a cognate single chain antibody (scFv). These cell-impermeant dyedrons exploit efficient intramolecular energy transfer from Cy3 donors to stoichiometrically amplify the fluorescence of MG chromophores that are activated by binding to the scFv. These chromophore enhancements, coupled with our optimized scFv, can significantly increase fluorescence emission generated by the dyedron/scFv complex to brightness levels several-fold greater than that for single fluorescent proteins and targeted small molecule fluorophores. Efficient intramolecular quenching of free dyedrons enables sensitive homogeneous (no wash) detection under typical tissue culture conditions, with undetectable nonspecific activation.
Szent-Gyorgyi
Christopher
C
Carnegie Mellon University, Molecular Biosensors and Imaging Center, Pittsburgh, Pennsylvania 15213, USA.
Schmidt
Brigitte F
BF
Fitzpatrick
James A J
JA
Bruchez
Marcel P
MP
D000199:Actins / Q000235:genetics / Q000378:metabolism; D002470:Cell Survival; D003116:Color; D003600:Cytosol / Q000378:metabolism; D050091:Dendrimers / Q000737:chemistry* / Q000378:metabolism*; D019020:Directed Molecular Evolution; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism*; D042541:Intracellular Space / Q000378:metabolism; D008853:Microscopy; D057054:Molecular Imaging; D010539:Permeability; D015202:Protein Engineering / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000166:cytology; D013194:Staining and Labeling
D000199:Actins; D050091:Dendrimers; D005456:Fluorescent Dyes