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Abstract <p><b>Objective:</b> The design of a new fluorogen-activating protein nano-frFAST and a series of fluorogens with an extended π-system are presented. <b>Methods:</b> 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 <i>in vitro</i> 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. <b>Results and Discussion:</b> We have shown that (<i>Z</i>)-5-((<i>E</i>)-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. <b>Conclusions:</b> 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.</p>

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

  • 期刊论文

  • 发表日期：

    2025年2月12日

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  • E. L. Sokolinskaya,
  • Yu. A. Bogdanova,
  • I. N. Myasnyanko,
  • A. I. Sokolov,
  • S. A. Krasnova,
  • M. S. Baranov

摘要

Abstract

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.