Base-type-selective high-resolution 13C edited NOESY for sequential assignment of large RNAs

Extensive spectral overlap presents a major problem for the NMR study of large RNAs. Here we present NMR techniques for resolution enhancement and spectral simplification of fully C-13 labelled RNA. High-resolution H-1-C-13 correlation spectra are obtained by combining TROSY-type experiments with multiple-band-selective homonuclear C-13 decoupling. An additional C-C filter sequence performs base-type-selective spectral editing. Signal loss during the filter is significantly reduced because of TROSY-type spin evolution. These tools can be inserted in any C-13-edited multidimensional NMR experiment. As an example we have chosen the C-13-edited NOESY which is a crucial experiment for sequential resonance assignment of RNA. Application to a 33-nucleotide RNA aptamer and a 76-nucleotide tRNA illustrates the potential of this new methodology.