title: Environment-sensing merocyanine dyes for live cell imaging applications.
abstract: Fluorescent biosensors based on environmentally sensitive dyes enable visualization and quantification of endogenous protein activation within living cells. Merocyanine dyes are especially useful for live cell imaging applications, as they are extraordinarily bright, have long wavelengths of excitation and emission, and can exhibit readily detectable fluorescence changes in response to environment. We sought to systematically examine the effects of structural features on key photophysical properties, including dye brightness, environmental responsiveness, and photostability, through the synthesis of a library of 25 merocyanine dyes, derived from combinatorial reaction of 5 donor and 5 acceptor heterocycles. Four of these dyes showed optimal properties for specific imaging applications and were subsequently prepared with reactive side chains and enhanced aqueous solubility using a one-pot synthetic method. The new dyes were then applied within a biosensor design for Cdc42 activation, where dye mero60 showed a remarkable 1470% increase in fluorescence intensity on binding activated Cdc42 in vitro. The dye-based biosensors were used to report activation of endogenous Cdc42 in living cells.
authors.affiliation: Department of Pharmacology, University of North Carolina, Chapel Hill, NC 27599, USA.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: MacNevin
authors.forename: Dmitriy
authors.initials: D
authors.lastname: Gremyachinskiy
authors.forename: Chia-Wen
authors.initials: CW
authors.lastname: Hsu
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.forename: Marie
authors.initials: M
authors.lastname: Rougie
authors.forename: Tamara T
authors.initials: TT
authors.lastname: Davis
authors.forename: Klaus M
authors.initials: KM
authors.lastname: Hahn
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000379:methods; D002470:Cell Survival; D004789:Enzyme Activation; D005456:Fluorescent Dyes / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D041681:NIH 3T3 Cells; D038761:Photobleaching; D011485:Protein Binding; D011744:Pyrimidinones / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D020764:cdc42 GTP-Binding Protein / Q000032:analysis* / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D011744:Pyrimidinones; C003954:merocyanine dye; D020764:cdc42 GTP-Binding Protein