3D C(CC)H TOCSY experiment for assigning protons and carbons in uniformly 13C- and selectively 2H-labeled RNA.

We have prepared RNAs uniformly 13C-labeled in the ribose ring and with the H3',H4',H5'/H5" protons specifically replaced with deuterons (3',4',5'/5"-2H-13C, termed d4-13C ribose), which offers the advantage of spectral simplification without sacrifice of sensitivity for the remaining protons. A 3D C(CC)H TOCSY experiment that uses a combination of cross-polarization transfer and deuterium decoupling improves the transfer of magnetization around the ribose ring and facilitates assignment of all ribose carbons and H1' and H2' protons in a 30-nucleotide RNA from HIV-2. These and other combinations of labeling and pulse sequence methodology should prove invaluable for the study of large RNAs, RNA-ligand, and RNA-protein complexes.