Nano-frFAST: Design of a New Genetically-Encoded Far-Red Fluorescent Label

Objective: The design of a new fluorogen-activating protein nano-frFAST and a series of fluorogens with an extended π-system are presented. Methods: The formation of imines of the corresponding cinnamaldehydes followed by [2 + 3]-cycloaddition was used for the synthesis of arylallylidene-imidazolones. The arylallylidene-rhodanine synthesis was performed by Knoevenagel condensation of the corresponding cinnamaldehydes with rhodanine. The other compounds were synthesized by condensation of arylidene-imidazolone analogues with isonicotinaldehyde. The new protein was screened in vitro and its optical properties were studied. Spectrofluorimetric titration was performed to evaluate the binding constant. Imaging of the [nano-frFAST–HPAR-DOM]-labeled cellular structures was performed by the wide-field fluorescence microscopy. Results and Discussion: We have shown that (Z)-5-((E)-3-(4-hydroxy-2,5-dimethoxyphenyl)allylidene)-2-thioxothiazolidin-4-one (hereafter, HPAR-DOM) can act as an excellent fluorogen for the nano-frFAST protein. We have demonstrated that the nano-frFAST–HPAR-DOM complex can be used in fluorescent labeling of individual compartments of the living HeLa Kyoto cells in the far-red region of the spectrum. Conclusions: We have developed the novel fluorogen-activating protein nano-frFAST and the fluorogen HPAR-DOM for this protein and demonstrated that this pair can be used as a genetically encoded far-red fluorescent label in living cells.