Characterization of benzopyrylium monomethine dyes as fluorescent probes for sensing and imaging of nucleic acids

Abstract

In this study, a series of benzopyrylium monomethine dyes bearing a benzothiazole moiety and mainly differing in their substitution pattern were characterized regarding their ability for nucleic acid binding and their use as fluorescent probes for sensing and imaging of nucleic acids. Dyes were characterized with special focus on structure and substituent effects on dye binding towards nucleic acids. Absorption maxima of the free, unbound dyes ranged from 474 to 498 nm. Fluorescence intensities of the unbound dyes were extremely low with emission maxima in the range of 512 to 584 nm depending on the polarity of the used solvent. Quantum yields of the free dyes were below 1 %. Upon binding to nucleic acids, however, dyes became highly fluorescent. Dyes with sterically demanding 2-di- or -triamino substituents exhibited a significant enhancement of the fluorescence quantum yield in presence of nucleic acids. The formation of stable dye-nucleic acid complexes was confirmed by time- and spectrally-resolved fluorescence measurements using a streak camera-based setup. Dye complexation was associated with prolonged lifetimes. All dyes were cell permeable and able to stain nucleic acids present in nuclear structures in living as well as in fixed cells. Colocalization analysis with nuclear specific reporters revealed that all dyes bind selectively to nucleic acids, which confirms their applicability as highly specific fluorescent probes for sensing nucleic acids and cell nuclei imaging. In comparison to commercially available nucleic acid stains, the investigated dyes displayed superior staining properties with high cell viability and less cytoplasmic enrichment. In conclusion, the results obtained by different complementary methods demonstrate that studied dyes are useful fluorescent probes for sensing and imaging of nucleic acids in-vitro, in fixed as well as in living cells. Bulkier dye substituents like piperazinyl and the presence of quaternary ammonium salts in the 2-amino side chain enhance nucleic acid binding significantly, thereby making the parent dye structure attractive for sensing applications and ongoing studies of structure–function relationship of nucleic acid stains.