Comparison of the solution structures of intramolecular DNA triple helices containing adjacent and non-adjacent CG·C⁺ triplets

The solution conformations of the intramolecular triple helices d(AGAAGA-X-TCTTCT-X-TC⁺TTC⁺T) and d(AAG-GAA-X-TTCCTT-X-TTC⁺C⁺TT) (X = non-nucleotide linker) have been determined by NMR. ¹H NMR spectra in H₂O showed that the third strand cytosine residues are fully paired with the guanine residues, each using two Hoogsteen hydrogen bonds. Determination of the ¹³C chemical shifts of the cytosine C6 and C5 and their one-bond coupling constants (¹J(CH)) conclusively showed that the Hoogsteen cytosine residues are protonated at N3. The global conformations of the two molecules determined with > 19 restraints per residue are very similar (RMSD = 0.96 Å). However, some differences in local conformation and dynamics were observed for the central two base triplets of the two molecules. The C N3H were less labile in adjacent CG·C⁺ triplets than in non-adjacent ones, indicating that the adjacent charge does not kinetically destabilize these triplets. The sugar conformations of the two adjacent cytosine residues were different and the 5'-residue was atypical of protonated cytosine. Hence, there are subtle effects of the interaction between two adjacent cytosine residues. The central two purines in each sequence showed non-standard backbone conformations, averaging between γ ≃ 60°and γ ≃= 180°. This may be related to the difference in the dependence of the thermodynamic stability on pH observed for these two sequences.