Free-probe fluorescence of light-up probes.
The fluorescence enhancement of light-up probes (thiazole orange (TO) conjugated peptide nucleic acids (PNAs)) upon hybridization to target nucleic acid depends on the probe sequence, mainly due to large variations in free-probe 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 fraction that has TO intramolecularly "back-bound" to the PNA bases. The intramolecular affinity constant for this unimolecular interaction was determined by temperature titrations using absorption spectroscopy, and the fluorescence quantum yields of the probes in back-bound conformation were calculated. The molar ratio of probes in back-bound conformation was 0.70-0.96 at 30 degrees C and 0.40-0.73 at 60 degrees C, and the fluorescence quantum yield in back-bound conformation varied between 0.0020 and 0.077 at 30 degrees C, and 0.00065-0.029 at 60 degrees C. These data show that the variation in free-probe fluorescence depends mainly on the fluorescence quantum yield of the probe in back-bound conformation and to a much lesser extent on the tendency of the probe to adopt the back-bound conformation. With increasing temperature the free-probe fluorescence decreases owing to both reduced degree of back-binding and a decrease of the fluorescence quantum yield in back-bound conformation.
Svanvik
N
N
Contribution from the Department of Molecular Biotechnology, Lundberg Institute and Department of Organic Chemistry, Chalmers University of Technology, P.O. Box 462, S-405 30 Göteborg, Sweden.
Nygren
J
J
Westman
G
G
Kubista
M
M
D001483:Base Sequence; D052160:Benzothiazoles; D005453:Fluorescence; D015345:Oligonucleotide Probes / Q000737:chemistry*; D020135:Peptide Nucleic Acids / Q000737:chemistry; D011804:Quinolines; D013816:Thermodynamics; D013844:Thiazoles / Q000737:chemistry
D052160:Benzothiazoles; D015345:Oligonucleotide Probes; D020135:Peptide Nucleic Acids; D011804:Quinolines; D013844:Thiazoles; C051000:thiazole orange