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

Abstract

Forced intercalation probes (FIT-probes) 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, 9 different TO derivatives were introduced into FIT probes. Fluorescence measurements in 6 different sequence 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 19-fold enhancement of fluorescence. Improved fluorescence quantum yields (Φds up to 0.53) and a high extinction coefficients (ε518 up to 91.000∙M−1∙cm−1) make this dye an interesting, and in some sequences superior alternative to the canonical thiazole orange used previously in FIT probes.