Solution structure of a DNA double helix incorporating four consecutive non-Watson-Crick base-pairs

A series of DNA 21-mers containing a variety of the 4 × 4 internal loop sequence 5′-CAAG-3′/3′-ACGT-5′ were studied using nuclear magnetic resonance (NMR) methodology and distance geometry (DG)/molecular dynamics (MD) approaches. Such oligomers exhibit excellent resolution in the NMR spectra and reveal many unusual NOEs (nuclear Overhauser effect) that allow for the detailed characterization of a DNA hairpin incorporating a track of four different non-Watson-Crick base-pairs in the stem. These include a wobble C·A base-pair, a sheared A·C base-pair, a sheared A·G base-pair, and a wobble G·T base-pair. Significantly different twisting angles were observed between the base-pairs in internal loop that results with excellent intra-strand and inter-strand base stacking within the four consecutive mismatches and the surrounding canonical base-pairs. This explains why it melts at 52°C even though five out of ten base-pairs in the stem adopt non-Watson-Crick pairs. However, the 4 × 4 internal loop still fits into a B-DNA double helix very well without significant change in the backbone torsion angles; only ζ torsion angles between the tandem sheared base-pairs are changed to a great extent from the gauche− domain to the trans domain to accommodate the cross-strand base stacking in the internal loop. The observation that several consecutive non-canonical base-pairs can stably co-exist with Watson-Crick base-pairs greatly increases the limited repertoire of irregular DNA folds and reveals the possibility for unusual structural formation in the functionally important genomic regions that have potential to become single-stranded.