Free-Probe Fluorescence of Light-up Probes

Abstract

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.

New Cyanine–Oligonucleotide Conjugates: Relationships between Chemical Structures and Properties

“…www.chemeurj.org ratio in conjugates 4, as previously reported in the case of PNA-4TO' conjugates. [37] ODNs 4 and 6 not only have the same base composition and sequence but also a very similar l max . The covalent attachment of the cyanines in conjugates 4 is adjacent to two cytosine residues whereas it is adjacent to two adenine residues for conjugates 6.…”

Quantitative Real-Time PCR Method for Detection of B-Lymphocyte Monoclonality by Comparison of κ and λ Immunoglobulin Light Chain Expression

“…Both probes are homopyrimidine sequences, which are known to exhibit very large signal enhancement on target binding (7 ). Both probes had the thiazole orange derivative, N-carboxypentyl-4-[(3Ј-methyl-1Ј,3Ј-benzothiazol-2Ј-yl)methylenyl]quinolinium iodide, as label attached to the peptide nucleic acid.…”

Noncovalent binding and fluorogenic response of cyanine dyes to DNA homoquadruplex and PNA-DNA heteroquadruplex structures

“…In our prior work, we used a covalently conjugated fluorogenic cyanine dye to provide a fluorescence response to PNA binding, as in the "light-up" probes originally reported by Ishiguro and coworkers for DNA 17 and subsequently by Svanvik and coworkers for PNA. 18,19 Recently, Ladame and coworkers 15 combined a PNA G 3 tract that could form one edge of a heteroquadruplex with an acridone stacking ligand previously shown to end-stack on G-tetrads. 20 Although the examples cited above clearly illustrate that a covalently conjugated dye can interact with a PNA-DNA heteroquadruplex, it was unclear to what extent the dye would interact with the heteroquadruplex if not for the covalent linkage to the PNA terminus.…”