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Conformation-Dependent Cleavage of Hairpin and Triplex Nucleic Acids by a Temperature-Insensitive Photonuclease

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journal contribution
posted on 19.09.1998, 00:00 by Suzanne A. Ciftan, H. Holden Thorp
Photolysis of Pt2(pop)44- in the presence of duplex DNA produces strand scission via abstraction of the 4‘- and 5‘-hydrogen atoms (pop = P2O5H22-). The cleavage intensities are higher for single-stranded DNA compared to those for duplex DNA because of lower electrostatic repulsion and greater solvent accessibility in single strands. When the single-stranded oligomer d(5‘-ACTGCCTTTTTGCTGAA) was photolyzed in the presence of Pt2(pop)44-, there was no significant or systematic change in cleavage intensity as a function of temperature in the range 28 to 98 °C. Cleavage of the hairpin d(5‘-ATCCTATTTTTTAGGAT) showed a higher cleavage intensity in the 5‘-TTTTT loop region compared to that for the base-paired nucleotides. Thermal denaturation of the hairpin gave a less selective cleavage pattern. The increase in cleavage at the duplex nucleotides could be used to estimate the Tm for the folded hairpin; thus, the cleavage pattern reflects the thermal denaturation of individual nucleotides. This concept was tested on the oligomer d(5‘-GAAGAGGTTTTTCCTCTTCTTTTTCTTCTCC), which exists in either a single-, double-, or triple-stranded form, depending on the pH and temperature. The folded forms exhibit higher reactivity in the loop regions compared to that for the base-paired nucleotides. Thermal denaturation of the base-paired nucleotides could be distinguished in the cleavage patterns and correlated with conventional optical absorption data. While optical melting curves provide greater precision in the observed Tm values, the cleavage intensity approach allows mapping of multiple transitions to individual nucleotides in the sequence. The combination of both approaches therefore offers a powerful method for following thermal denaturation of DNA at A, T, and C nucleotides.