TRIPLE-HELIX DNA OLIGOMER MELTING MEASURED BY FLUORESCENCE POLARIZATION ANISOTROPY

A synthetic DNA triple helix sequence was formed by annealing a pyrimi dinic 21 mer single strand se quence onto the complementary purinic se quence centred on a 27 mer duplex DNA. Melting of the third strand was monitored by UV spectrophotometry in the temperature range 10-90 degr ees C. The T-m of the tripler, 37 degrees C, was well separated from t he onset of duplex melting, When the same triple helix was formed on t he duplex bearing one nick in the center of the pyrimidinic sequence t he T-m of the triplex was shifted to approximately 32 degrees C and ov erlapped the melting of the duplex. We have used fluorescence polariza tion anisotropy (FPA) measurements of ethidium bromide (EB) intercalat ed in duplex and tripler samples to determine the hydrodynamic paramet ers in the temperature range 10-40 degrees C. The fluorescence lifetim e of EB in the samples of double and triple stranded DNA is the same ( 21.3+/-0.5 ns) at 20 degrees C, indicating that the geometries of the intercalation sites are similar. The values for the hydration radii of the duplex, normal tripler, and nicked triplex samples were 10.7+/-0. 2, 12.2+/-0.2, and 12.0+/-0.2 Angstrom. FPA measurements on normal tri pler DNA as a function of temperature gave a melting profile very simi lar to that derived by UV absorption spectroscopy. For the tripler car rying a nick, the melting curve obtained using FPA showed a clear shif t compared with that obtained for the normal tripler sample. The torsi onal rigidity of the tripler forms was found to be higher than that of the duplex form.