High resolution 4D HPCH experiment for sequential assignment of (13)C-labeled RNAs via phosphodiester backbone

The three-dimensional structure determination of RNAs by NMR spectroscopy requires sequential resonance assignment, often hampered by assignment ambiguities and limited dispersion of (1)H and (13)C chemical shifts, especially of C4′/H4′. Here we present a novel through-bond 4D HPCH NMR experiment involving phosphate backbone where C4′–H4′ correlations are resolved along the (1)H3′–(31)P spectral planes. The experiment provides high peak resolution and effectively removes ambiguities encountered during assignments. Enhanced peak dispersion is provided by the inclusion of additional (31)P and (1)H3′ dimensions and constant-time evolution of chemical shifts. High spectral resolution is obtained by using non-uniform sampling in three indirect dimensions. The experiment fully utilizes the isotopic (13)C-labeling with evolution of C4′ carbons. Band selective (13)C inversion pulses are used to achieve selectivity and prevent signal dephasing due to the C4′–C3′ and C4′–C5′ homonuclear couplings. Multiple quantum line narrowing is employed to minimize sensitivity loses. The 4D HPCH experiment is verified and successfully applied to a non-coding 34-nt RNA consisting typical structure elements and a 14-nt RNA hairpin capped by cUUCGg tetraloop.