Intramolecular Dimers:  A New Strategy to Fluorescence Quenching in Dual-Labeled Oligonucleotide Probes

• Published in: Journal of the American Chemical Society 2002-06, Vol.124 (24), p.6950-6956
• Main Authors: Johansson, Mary Katherine, Fidder, Henk, Dick, Daren, Cook, Ronald M
• Format: Article
• Language: English
• Subjects: Actins - genetics; Atomic and molecular physics; DNA, Complementary - analysis; DNA, Complementary - chemistry; Exact sciences and technology; Fluorescence; Fluorescence and phosphorescence; radiationless transitions, quenching (intersystem crossing, internal conversion); Fluorescent Dyes - chemistry; Molecular properties and interactions with photons; Oligonucleotide Probes - chemistry; Physics; Radiationless transitions, quenching
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja025678o

Many genomics assays use profluorescent oligonucleotide probes that are covalently labeled at the 5‘ end with a fluorophore and at the 3‘ end with a quencher. It is generally accepted that quenching in such probes without a stem structure occurs through Förster resonance energy transfer (FRET or FET) and that the fluorophore and quencher should be chosen to maximize their spectral overlap. We have studied two dual-labeled probes with two different fluorophores, the same sequence and quencher, and with no stem structure:  5‘Cy3.5−β-actin−3‘BHQ1 and 5‘FAM−β-actin−3‘BHQ1. Analysis of their absorption spectra, relative fluorescence quantum yields, and fluorescence lifetimes shows that static quenching occurs in both of these dual-labeled probes and that it is the dominant quenching mechanism in the Cy3.5−BHQ1 probe. Absorption spectra are consistent with the formation of an excitonic dimer, an intramolecular heterodimer between the Cy3.5 fluorophore and the BHQ1 quencher.