Abstract
Fluorogens are an attractive type of dye for imaging applications, eliminating time-consuming washout steps from staining protocols. With just a handful of reported fluorogen-protein pairs, mostly in the green region of spectra, there is a need for the expansion of their spectral range. Still, the origins of solvatochromic and fluorogenic properties of the chromophores suitable for live-cell imaging are poorly understood. Here we report on the synthesis and labeling applications of novel red-shifted fluorogenic cell-permeable green fluorescent protein (GFP) chromophore analogs.
Highlights
Fluorogenic dyes are compounds which are non-fluorescent by themselves in solution but become highly emissive in a complex with the target molecule
We report on the synthesis and labeling applications of novel red-shifted fluorogenic cell-permeable green fluorescent protein (GFP) chromophore analogs
Widefield fluorescence microscopy for measurement of background fluorescence and membrane fluorescence of fluorogenic dye was performed with the BZ-9000 fluorescence microscope (Keyence) equipped with 40× dry and 63× oil-immersion objectives
Summary
Fluorogenic dyes are compounds which are non-fluorescent by themselves in solution but become highly emissive in a complex with the target molecule. The list of target molecules compatible with cellular imaging applications includes RNA aptamers [1], and various proteins [2,3,4,5]. These fluorogen–target pairs hold the promise for simple no-washout cellular staining, an increase in the signal-to-noise ratio, and photostability in cellular imaging applications. IInnoouurrpprerevvioiouusswworokr,kw, we deedveevloeploepdepdroptreointehinoshtsosctaspcaabpleaboflereocforveecroinvgertihnegfltuhoerflesucoernecseceonf csuecohf cshurcohmcohprohmoroepsh(oprreism(aprriilmy acroilmy pcoomunpdouMnd73M9 7[349], [S4]c,hSecmheem1e),1a),nadnddedmemonosntsratrtaetdedththatatrreessttrricicttioionn ooff fflleexxiibbiilliittyy iiss nnoott tthhee oonnllyy wwaayy ttoo iinndduuccee fflluuoorrooggeenniicc bbeehhaavviioorr. IInn tthhiiss wwoorrkk,, wwee eexxpplloorree aa ppaanneell ooff MM773399 ddeerriivvaattiivveess ((SScchheemmee 11)),, aaiimmiinngg ttoo ccrreeaattee pprrootteeiinn––ddyyee bbiippaarrttiittee ttaaggss wwiitthh rreedd--sshhiifftteedd fflluuoorreesscceennccee
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