C4′/H4′ selective, non-uniformly sampled 4D HC(P)CH experiment for sequential assignments of (13)C-labeled RNAs

A through bond, C4′/H4′ selective, “out and stay” type 4D HC(P)CH experiment is introduced which provides sequential connectivity via H4′((i))–C4′((i))–C4′((i−1))–H4′((i−1)) correlations. The (31)P dimension (used in the conventional 3D HCP experiment) is replaced with evolution of better dispersed C4′ dimension. The experiment fully utilizes (13)C-labeling of RNA by inclusion of two C4′ evolution periods. An additional evolution of H4′ is included to further enhance peak resolution. Band selective (13)C inversion pulses are used to achieve selectivity and prevent signal dephasing due to the of C4′–C3′ and C4′–C5′ homonuclear couplings. For reasonable resolution, non-uniform sampling is employed in all indirect dimensions. To reduce sensitivity losses, multiple quantum coherences are preserved during shared-time evolution and coherence transfer delays. In the experiment the intra-nucleotide peaks are suppressed whereas inter-nucleotide peaks are enhanced to reduce the ambiguities. The performance of the experiment is verified on a fully (13)C, (15)N-labeled 34-nt hairpin RNA comprising typical structure elements. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10858-014-9861-z) contains supplementary material, which is available to authorized users.