Parallel-stranded duplex DNA containing blocks of trans purine-purine and purine-pyrimidine base pairs

Abstract

A 30 base pair parallel-stranded (ps) duplex ps-L1, L2 composed of two adjoined purine-purine and purine-pyrimidine sequence blocks has been characterized thermodynamically and spectroscopically. The 5′-terminal 15 residues in both strands (‘left-half’) consisted of the alternating d(GA) ₇ G sequence that forms a ps homoduplex secondary structure stabilized by d(G-G) and d(A-A) base pairs. The 3′-terminal 15 positions of the sequence (‘right-half’') were combinations of A and T with complementary reverse Watson-Crick d(A-T) base pairing between the two strands. The characteristics of the full length duplex were compared to those of the constituent left and right halves in order to determine the compatibility of the two ps helical forms. The thermal denaturation curves and hyperchromicity profiles of all three duplexes determined by UV absorption spectroscopy were characteristic of ps-DNA, in accordance with previous studies. The thermodynamic properties of the 30 bp duplex corresponded within experimental error to the linear combination of the two 15-mers. Thus, the T _m and AH _vH of ps-L1L2 in 10 mM MgCI ₂ , derived from analyses according to a statistical mechanical formulation for the helix - coil transition, were 43°C and 569 kJ mol−1, compared to 21 °C, 315 kJ mol−1(ps-F5-F6) and 22°C, 236 kJ mol˜1 (ps-GA ₁₅ ). The UV absorption and CD spectra of ps-L1 · L2 and the individual 15-mer ps motifs were also compared quantitatively. The sums of the two constituent native spectra (left + right halves) accurately matched that of the 30 bp duplex, with only small deviations in the 195 − 215 nm (CD) and 220 − 240 nm (absorption) regions. Based on analysis by native gel electrophoresis, the sequences studied formed duplex structures exclusively; there were no indications of higher order species. Chemical modification with diethyl pyrocarbonate showed no hyperreactivity of the junctional bases, indicating a smooth transition between the two parallel-stranded conformations. We conclude that under given salt conditions