Intramolecular dimers: a new strategy to fluorescence quenching in dual-labeled oligonucleotide probes.
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.
Johansson
Mary Katherine
MK
Biosearch Technologies, 81 Digital Drive, Novato, California 94949, USA. marykat@biosearchtech.com
Fidder
Henk
H
Dick
Daren
D
Cook
Ronald M
RM
D000199:Actins / Q000235:genetics; D018076:DNA, Complementary / Q000032:analysis / Q000737:chemistry; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D015345:Oligonucleotide Probes / Q000737:chemistry*
D000199:Actins; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes