STABILITY, SPECIFICITY AND FLUORESCENCE BRIGHTNESS OF MULTIPLY-LABELED FLUORESCENT DNA PROBES

In this work, we studied the fluorescence and hybridization of multipl y-labeled DNA probes which have the hydrophilic fluorophore -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 intensit y, 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 i ntensity and Phi(f) are maximized when labeling every 6th base using t he long linker, With a less hydrophilic dye, a labeling density this h igh could not be achieved without serious quenching of the fluorescenc e, The target specificity of multiply-labeled DNA probes was just as h igh as compared to the unmodified control probe, however, a less stabl e probe-target duplex is formed that exhibits a lower melting temperat ure, A mechanism that accounts for this destabilization is proposed wh ich is consistent with our data, It involves dye-dye and dye-nucleotid e interactions which appear to stabilize a single-stranded conformatio n of the probe.