UNAMBIGUOUS STRUCTURE CHARACTERIZATION OF A DNA-RNA TRIPLE-HELIX BY N-15-FILTERED AND C-13-FILTERED NOESY SPECTROSCOPY

DNA . DNARNA triple helices of the pyrimidine . purine*pyrimidine mot if (where . indicates Watson-Crick pairing and indicates Hoogsteen p airing) appear to be very stable, which has important implications for the development of novel antisense strategies. Here we present the fi rst structural NMR studies on such a system, composed of a DNA hairpin with a homopurine-homopyrimidine stem sequence and a single-stranded RNA oligonucleotide containing exclusively pyrimidine residues. In the se investigations an unlabeled DNA hairpin and a uniformly C-13/N-15-e nriched RNA oligonucleotide were utilized in combination with X-edited H-1 NMR spectroscopy. Improved N-15 (omega(2)) filtered NOESY and C-1 3 (omega(1)) filtered NOESY are presented by which we were able to dif ferentiate between intrastrand, i.e., DNA-DNA and RNA-RNA, and interst rand, i.e., DNA-RNA, NOE contacts. It is unambiguously established tha t the complex forms a right-handed triple helix, with the RNA strand s ituated in the major groove of the Watson-Crick stem of the hairpin. T he interaction is stabilized by the formation of Hoogsteen-type base p airs between the RNA strand and the purine strand of the DNA. These st rands run parallel to each other. The characterization of the DNA-RNA triple helix structure described here shows that this type of experime nt forms a valuable instrument in the structure determination of bimol ecular systems of nucleic acids.