Structure and folding dynamics of a DNA hairpin with a stabilising d(GNA) trinucleotide loop: influence of base pair mis-matches and point mutations on conformational equilibria

Abstract

Hairpins are known to play specific roles in DNA– and RNA–protein recognition. Various disease states are thought to originate from the ill-timed formation of a hairpin loop during transcription, particularly in the context of triplet repeats which are associated with myotonic dystrophy, fragile X syndrome and other genetic disorders. An understanding of nucleic acid folding mechanisms requires a detailed appreciation of the timescales of these local folding events, a characterisation of the conformational equilibria that exist in solution and the influence of point mutations on the relative stabilities of the different species. We investigate using NMR and CD spectroscopy the structure and dynamics of a DNA hairpin containing a highly stabilising cGNAg loop. The single-stranded 13-mer 5′-d(GCTACGNAGTCGC) with N = T folds to form a hairpin structure which accommodates a C–T mis-matched base pair within the double-stranded stem region. The hairpin is in equilibrium with a double-stranded duplex form with the mixture of two interconverting conformations in slow exchange on the NMR timescale (1–2 s^–1 at 308 K). We are able to characterise the dynamics of the interconversion process by NMR magnetisation transfer and by CD stopped-flow kinetic experiments. The latter shows that the hairpin folds too rapidly to detect by this method (>500 s^–1) and forms in a “kinetic overshoot” followed by a much slower equilibration to a mixture of conformations (∼0.13 s^–1 at 298 K). A point mutation that converts the GTA to a GAA loop sequence destabilises the intermolecular duplex structure and enables us to unambiguously assign the various dynamic processes that are taking place.