New 2D and 3D IH-13C-15N triple resonance experiments are presented which allow unambiguous assignments of intranucleotide HI'-H8(H6) connectivities in 13C-and 15N-labeled RNA oligonucleotides. Two slightly different experiments employing double INEPT forward and back coherence transfers are optimized to obtain the HI'-CI'-Ng/N1 and H8/H6-C8/C6-Ng/N1 connectivities, respectively. The correlation of HI' protons to glycosidic nitrogens Ng/N1 is obtained in a nonselective fashion. To correlate H8/H6 with their respective glycosidic nitrogens, selective 13C-refocusing and 15N-inversion pulses are applied to optimize the magnetization transfers along the desired pathway. The approach employs the heteronuclear one-bond spin-spin interactions and allows the 2D ~ H-ISN and 3 D ~ H-~3C-15N chemical shift correlation of nuclei along and adjacent to the glycosidic bond. Since the intranucleotide correlations obtained are based exclusively on through-bond scalar interactions, these experiments resolve the ambiguity of intra-and internucleotide HI'-H8(H6) assignments obtained from the 2D NOESY spectra. These experiments are applied to a 30-base RNA oligonucleotide which contains the binding site for Rev protein from HIV.
Until recently, NMR studies of nucleic acids have been largely limited to 1H and some 31p experiments. With the recent advent of methods for preparing uniformly labeled RNA oligonucleotides by enzymatic synthesis (Milligan et al., 1987) from 15N-and/or 13C-labeled NTPs isolated from E. coli (Nikonowicz et al., 1992;Michnicka et al., 1993) or methanolotrophic bacteria (Batey et al., 1992) grown on labeled media, it is now possible to adapt the heteronuclear multidimensional NMR methods developed for the study of protein structures (Kay et al., 1990;Clore and Gronenborn, 1991;Bax and Grzesiek, 1993) to the special problems of RNA oligonucleotides. Several such double resonance experiments have been reported, taking advantage of the *To whom correspondence should be addressed.