title: Novel DNA probes with low background and high hybridization-triggered fluorescence.
abstract: Novel fluorogenic DNA probes are described. The probes (called Pleiades) have a minor groove binder (MGB) and a fluorophore at the 5'-end and a non-fluorescent quencher at the 3'-end of the DNA sequence. This configuration provides surprisingly low background and high hybridization-triggered fluorescence. Here, we comparatively study the performance of such probes, MGB-Eclipse probes, and molecular beacons. Unlike the other two probe formats, the Pleiades probes have low, temperature-independent background fluorescence and excellent signal-to-background ratios. The probes possess good mismatch discrimination ability and high rates of hybridization. Based on the analysis of fluorescence and absorption spectra we propose a mechanism of action for the Pleiades probes. First, hydrophobic interactions between the quencher and the MGB bring the ends of the probe and, therefore, the fluorophore and the quencher in close proximity. Second, the MGB interacts with the fluorophore and independent of the quencher is able to provide a modest (2-4-fold) quenching effect. Joint action of the MGB and the quencher is the basis for the unique quenching mechanism. The fluorescence is efficiently restored upon binding of the probe to target sequence due to a disruption in the MGB-quencher interaction and concealment of the MGB moiety inside the minor groove.
authors.affiliation: Nanogen, Inc., 21720 23rd Drive SE, Suite 150, Bothell, WA 98021, USA. elukhtanov@nanogen.com
authors.forename: Eugeny A
authors.initials: EA
authors.lastname: Lukhtanov
authors.forename: Sergey G
authors.initials: SG
authors.lastname: Lokhov
authors.forename: Vladimir V
authors.initials: VV
authors.lastname: Gorn
authors.forename: Mikhail A
authors.initials: MA
authors.lastname: Podyminogin
authors.forename: Walt
authors.initials: W
authors.lastname: Mahoney
mesh_terms: D015342:DNA Probes / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D013696:Temperature
chemical_list: D015342:DNA Probes; D005456:Fluorescent Dyes