Analysis of GAA/TTC DNA triplexes using nuclear magnetic resonance and electrospray ionization mass spectrometry

Abstract

The formation of a GAA/TTC DNA triplex has been implicated in Friedreich’s ataxia. The destabilization of GAA/TTC DNA triplexes either by pH or by binding to appropriate ligands was analyzed by nuclear magnetic resonance (NMR) and positive-ion electrospray mass spectrometry. The triplexes and duplexes were identified by changes in the NMR chemical shifts of H8, H1, H4, 15N7, and 15N4. The lowest pH at which the duplex is detectable depends upon the overall stability and the relative number of Hoogsteen C∘G to T∘A basepairs. A melting pH (pH m) of 7.6 was observed for the destabilization of the (GAA) 2T 4(TTC) 2T 4(CTT) 2 triplex to the corresponding Watson–Crick duplex and the T 4(CTT) 2 overhang. The mass spectrometric analyses of (TTC) 6•(GAA) 6∘(TTC) 6 triplex detected ions due to both triplex and single-stranded oligonucleotides under acidic conditions. The triplex ions disappeared completely at alkaline pH. Duplex and single strands were detectable only at neutral and alkaline pH values. Mass spectrometric analyses also showed that minor groove-binding ligands berenil, netropsin, and distamycin and the intercalating ligand acridine orange destabilize the (TTC) 6•(GAA) 6∘(TTC) 6 triplex. These NMR and mass spectrometric methods may function as screening assays for the discovery of agents that destabilize GAA/TTC triplexes and as general methods for the characterization of structure, dynamics, and stability of DNA and DNA–ligand complexes.