Free-probe fluorescence of light-up probes

The fluorescence enhancement of light-up probes (thiazole orange (TO) conju gated peptide nucleic acids (PNAs)) upon hybridization to target nucleic ac id depends on the probe sequence, mainly due to large variations in free-pr obe fluorescence. Here we study three probes where the fluorescence in free state varies more than 50-fold. We find that this variation is due to a fr action that has TO intramolecularly "back-bound" to the PNA bases. The intr amolecular affinity constant for this unimolecular interaction was determin ed by temperature titrations using absorption spectroscopy, and the fluores cence quantum yields of the probes in back-bound conformation were calculat ed. The molar ratio of probes in back-bound conformation was 0.70-0.96 at 3 0 degreesC and 0.40-0.73 at 60 degreesC, and the fluorescence quantum yield in back-bound conformation varied between 0.0020 and 0.077 at 30 degreesC, and 0.00065-0.029 at 60 degreesC. These data show that the variation in fr ee-probe fluorescence depends mainly on the fluorescence quantum yield of t he probe in back-bound conformation and to a much lesser extent on the tend ency of the probe to adopt the back-bound conformation. With increasing tem perature the free-probe fluorescence decreases owing to both reduced degree of back-binding and a decrease of the fluorescence quantum yield in back-b ound conformation.