Spectral and physical characterization of the inverted terminal repeat DNAstructure from adenoassociated virus 2

An oligodeoxynucleotide (ODN) that includes elements found in secondary str uctures at the 5'- and 3'-ends of adenoassociated virus 2 virion DNA was sy nthesized by ligation of three overlapping ODNs, The most stable secondary structure was calculated to be branched, with a 61 bp duplex stem, terminat ing in a three-way junction with 9 bp arms. The electrophoretic mobility of the ODN is slower than expected for normal duplex DNA of the same size, su ggesting a bent or branched conformation. CD spectra indicate that the ITR structure is largely B form DNA, although there is a slight blue shift comp ared to the spectra of the isolated stem and loop elements, Thermal melting experiments indicate that the hairpin is significantly more stable than th e isolated stem and loop elements. Singular value decomposition of UV spect ra obtained as a function of temperature indicates that four species contri bute to changes in the spectra upon denaturation, indicating that the melti ng is not a simple two-state process. Characterization of the branched ODN by differential scanning calorimetry permits estimation of the enthalpy of melting by a model-free analysis, yielding Delta H-cal = 614 kcal mol(-1). This value agrees with the enthalpy computed for the most stable secondary structure.