Enhanced photostability of genetically encodable fluoromodules based on fluorogenic cyanine dyes and a promiscuous protein partner.
Fluoromodules are discrete complexes of biomolecules and fluorogenic dyes. Binding of the dyes to their cognate biomolecule partners results in enhanced dye fluorescence. We exploited a previously reported promiscuous binding interaction between a single-chain, variable fragment antibody protein and a family of cyanine dyes to create new protein-dye fluoromodules that exhibit enhanced photostability while retaining high affinity protein-dye binding. Modifications to the dye structure included electron-withdrawing groups that provide resistance to photo-oxidative damage. Low nanomolar equilibrium dissociation constants were found for the new dyes. Fluorescence microscopy illustrates how yeast can be surface-labeled with three different colors based on a single protein and appropriately chosen dyes.
Shank
Nathaniel I
NI
Department of Chemistry, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, USA.
Zanotti
Kimberly J
KJ
Lanni
Frederick
F
Berget
Peter B
PB
Armitage
Bruce A
BA
D001666:Binding Sites, Antibody; D002232:Carbocyanines / Q000032:analysis / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism*; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D015394:Molecular Structure; D010777:Photochemistry; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000166:cytology
D002232:Carbocyanines; D005456:Fluorescent Dyes; D007140:Immunoglobulin Fab Fragments