TEMPERATURE END QUENCHING STUDIES OF FLUORESCENCE POLARIZATION DETECTION OF DNA HYBRIDIZATION

The effects of temperature and collisional quenching on fluorescence p olarization detection of DNA hybridization were studied using measurem ents of fluorescence intensity and anisotropy and the dynamic decay of these properties. Three different tethers, 3, 6, and 12 carbons in le ngth, were used to attach fluorescein label to the 5' end of the 33-ba se oligomers. Perrin plots showed that the effective rotating volume d ecreases with increasing tether length and approximately doubles upon hybridization. Hybridization increases the association between the tet hered dye and the DNA for the shorter tethers but displaces the fluore scein on the 12C tether from the DNA, forcing it into greater contact with the bulk solution. The 6C tether appears to promote sequence-spec ific interaction between fluorescein label and the oligomer, which cau sed unexpectedly protection from collisional quenching. In all cases, there appear to exist several possible conformations for the tethered fluorescein. As temperature is increased, these conformations tend to collapse into a single, average or preferred, conformation. The result s demonstrate the importance of the selection of tether, dye, and DNA probe in designing a polarization strategy for detection of DNA hybrid ization, particularly with respect to tether length and DNA probe sequ ence.