Triple helix DNA oligomer melting measured by fluorescence polarization anisotropy

A synthetic DNA triple helix sequence was formed by annealing a pyrimidinic 21 mer single strand se quence onto the complementary purinic sequence centred on a 27 mer duplex DNA. Melting of the third strand was monitored by UV spectrophotometry in the temperature range 10-90 degrees C. The T-m of the tripler, 37 degrees C, was well separated from the onset of duplex melting, When the same triple helix was formed on the duplex bearing one nick in the center of the pyrimidinic sequence the T-m of the triplex was shifted to approximately 32 degrees C and overlapped the melting of the duplex. We have used fluorescence polarization anisotropy (FPA) measurements of ethidium bromide (EB) intercalated in duplex and tripler samples to determine the hydrodynamic parameters in the temperature range 10-40 degrees C. The fluorescence lifetime 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 tripler DNA as a function of temperature gave a melting profile very similar to that derived by UV absorption spectroscopy. For the tripler carrying a nick, the melting curve obtained using FPA showed a clear shift compared with that obtained for the normal tripler sample. The torsional rigidity of the tripler forms was found to be higher than that of the duplex form.