Coumarin amine-reactive dye C392STP: an efficient building block to synthesize single-labeled oligonucleotides with application as FISH probes

Abstract

The demand of new reactive fluorophores with improved photophysical characteristics for labeling oligonucleotide probes is growing. Sodium (E/Z)-4-(4-(2-(6,7-dimethoxycoumarin-3-yl)vinyl)-benzoyl)- 2,3,5,6-tetrafluoro-benzenesulfonate (C392STP) is an amine-reactive fluorescent dye that has recently shown interesting photophysical properties. In this work, we investigate the possibility of: i) synthesizing it cheaply and easily; ii) using this reactive dye to synthesize single-labeled fluorescent oligonucleotides; and iii) applying the resulting fluorescently labeled oligonucleotides as fluorescence in situ hybridization (FISH) probes. According to the results obtained in this study, C392STP: i) can be synthesized through a low cost and simple pathway; ii) is an efficient building block for mono-labeling of oligomers with an efficiency as high as that of an amine-reactive Alexa Fluor® 488 dye supplied in a commercial oligonucleotide labeling kit. Furthermore, the RNA-FISH assays revealed that C392-mono-labeled probes containing standard carbon linkers (C6) exhibited effective performance as RNA-FISH probes. These findings evidenced the applicability of C392STP in labeling of biomolecules and bioimaging. The results invite the researchers to continue exploring other uses of this reactive dye for exploiting its potential in biological applications.