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-(epsilon-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 (Phi(f)), probe-target stability and specificity, For the hydrophilic dye Cy3, we have demonstrated that the fluorescence intensity and Phi(f) are 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.