Three-Dimensional Homonuclear NOESY-TOCSY of an Intramolecular Pyrimidine-Purine-Pyrimidine DNA Triplex Containing a Central G∙TA Triple: Nonexchangeable Proton Assignments and Structural Implications

Two-and three-dimensional homonuclear ¹H NMR spectroscopic techniques have been applied to obtain nearly complete nonexchangeable proton assignments for a 31-residue intramolecular pyrimidine-purine-pyrimidine DNA triplex containing a central G∙TA triple in D₂O. An assignment strategy for obtaining resonance assignments for DNA protons from a 3D NOESY-TOCSY spectrum is proposed. The strategy utilizes the H1ʹ/H5 ω₃planes and relies on the recognition of cross-peak patterns for obtaining both intraresidue as well as sequential assignments. On the basis of the cross-peaks observed in the 2D and 3D spectra, a few structural features of the triplex have been delineated qualitatively. All three strands of the triplex adopt a right-handed helical conformation, and, despite the introduction of a central purine guanosine, there is no evidence for major structural distortions in the protonated third strand on the basis of a qualitative interpretation of NMR data. Several interstrand contacts between the purine and the Hoogsteen pyrimidine strands are observed which define the relative orientation of the bases and sugars in these two strands. The presence of strong NOEs between the methyl protons of thymine and the H1ʹ proton of guanosine defines the preferred base-pairing alignment of guanosine at the G∙TA triple site. The general approaches illustrated in this study extend the range of DNA molecules accessible for detailed structural investigation by high-resolution NMR spectroscopy.