New Thiazole Orange Derivatives for Improved Fluorescence Signaling of DNA FIT Probes

Forced intercalation probes (FIT probes) are nucleic acid probes in which an intercalator dye of the thiazole orange (TO) family serves as a surrogate nucleobase. Hybridization of FIT probes is accompanied by enhancements of fluorescence. Looking for ways to increase turn-on and brightness of fluorescence, we herein report the synthesis of new fluorogenic base surrogates. In total, nine different TO derivatives were introduced into FIT probes. Fluorescence measurements in six different sequences revealed that substitution at both the quinoline and the benzothiazole part affects fluorescence turn-on upon hybridization and brightness of probe-target duplexes. A TO derivative containing a tricyclic benzothiazole provided FIT probes signaling hybridization by up to 18.6-fold enhancement of fluorescence. Improved fluorescence quantum yields (Phi(ds) up to 0.53) and high extinction coefficients (epsilon(518) up to 91000 M-1 center dot cm(-1)) make this dye an interesting, and in some sequences superior, alternative to the canonical thiazole orange used previously in FIT probes.

Automated summary

Forced intercalation probes (FIT probes) are nucleic acid probes with a fluorescent dye surrogate nucleobase that enhances fluorescence upon hybridization. We synthesized new fluorogenic base surrogates by introducing nine different derivatives into FIT probes. Substituents at the quinoline and benzothiazole parts of the probes affect fluorescence turn-on and brightness of probe-target duplexes. A tricyclic benzothiazole derivative showed up to 18.6-fold enhancement of fluorescence upon hybridization, making it a superior alternative to thiazole orange in FIT probes.