title: Intramolecular dimers: a new strategy to fluorescence quenching in dual-labeled oligonucleotide probes.
abstract: 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-beta-actin-3'BHQ1 and 5'FAM-beta-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.
authors.affiliation: Biosearch Technologies, 81 Digital Drive, Novato, California 94949, USA. marykat@biosearchtech.com
authors.forename: Mary Katherine
authors.initials: MK
authors.lastname: Johansson
authors.forename: Henk
authors.initials: H
authors.lastname: Fidder
authors.forename: Daren
authors.initials: D
authors.lastname: Dick
authors.forename: Ronald M
authors.initials: RM
authors.lastname: Cook
mesh_terms: D000199:Actins / Q000235:genetics; D018076:DNA, Complementary / Q000032:analysis / Q000737:chemistry; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D015345:Oligonucleotide Probes / Q000737:chemistry*
chemical_list: D000199:Actins; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes