Stability, specificity and fluorescence brightness of multiply-labeled fluorescent DNA probes.
In this work, we studied the fluorescence and hybridization of multiply-labeled DNA probes which have the hydrophilic fluorophore 1-(straightepsilon-carboxypentynyl)-1'-ethyl- 3,3,3', 3'-tetramethylindocarbocyanine-5,5'-disulfonate (Cy3) attached via either a short or long linker at the C-5 position of deoxyuridine. We describe the effects of labeling density, fluorophore charge and linker length upon five properties of the probe: fluorescence intensity, the change in fluorescence upon duplex formation, the quantum yield of fluorescence (Phif), probe-target stability and specificity. For the hydrophilic dye Cy3, we have demonstrated that the fluorescence intensity andPhifare maximized when labeling every 6th base using the long linker. With a less hydrophilic dye, a labeling density this high could not be achieved without serious quenching of the fluorescence. The target specificity of multiply-labeled DNA probes was just as high as compared to the unmodified control probe, however, a less stable probe-target duplex is formed that exhibits a lower melting temperature. A mechanism that accounts for this destabilization is proposed which is consistent with our data. It involves dye-dye and dye-nucleotide interactions which appear to stabilize a single-stranded conformation of the probe.
Randolph
J B
JB
Center for Light Microscope Imaging and Biotechnology and Department of Chemistry, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, USA. jr4h@andrew.cmu.edu
Waggoner
A S
AS
D002232:Carbocyanines / Q000737:chemistry*; D015342:DNA Probes; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D015394:Molecular Structure; D012680:Sensitivity and Specificity
D002232:Carbocyanines; D015342:DNA Probes; D005456:Fluorescent Dyes; C089187:cyanine dye 3