Structural characterisation of a uracil containing hairpin DNA by NMR and molecular dynamics

Abstract

Three-dimensional (3D) structure of a hairpin DNA d-CTAGAGGATCCTTTUGGATCCT (22mer; abbreviated as U4-hairpin), which has a uracil nucleotide unit at the fourth position from the 5' end of the tetraloop has been solved by NMR spectroscopy. The ¹H resonances of this hairpin have been assigned almost completely. NMR restrained molecular dynamics and energy minimisation procedures have been used to describe the 3D structure of the U4 hairpin. This study establishes that the stem of the hairpin adopts a right handed B-DNA conformation while the T₁₂ and U₁₅ nucleotide stack upon 3' and 5' ends of the stem, respectively. Further, T₁₄ stacks upon both T₁₂ and U₁₅ while T₁₃ partially stacks upon T₁₄. Very weak stacking interaction is observed between T₁₃ and T₁₂. All the individual nucleotide bases adopt 'anti' conformation with respect to their sugar moiety. The turning phosphate in the loop is located between T₁₃ and T₁₄. The stereochemistry of U15 mimics the situation where uracil would stack in a B-DNA conformation. This could be the reason as to why the U4-hairpin is found to be the best substrate for its interaction with uracil DNA glycosylase (UDG) compared to the other substrates in which the uracil is at the first, second and third positions of the tetra-loop from its 5' end, as reported previously.