Improved sensitivity and resolution in 1H-13C NMR experiments of RNA

NMR studies of the structure and dynamics in RNA are greatly facilitated by the use of C-13-labeled molecules and H-1-C-13 correlation experiments. We demonstrate here that spin-state selective C-13 frequency editing yields greatly improved sensitivity and resolution in H-1-C-13 correlation spectra of RNA. A sensitivity enhanced version of the previously introduced TROSY sequence (Pervushin et al., Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 12366-12371) is proposed, which offers a root 2 signal enhancement. When compared to a sensitivity enhanced HSQC sequence an up to 3-fold increase in both the sensitivity and resolution is observed for the base carbons in a 15% C-13-labeled 33-mer RNA at a magnetic field strength of 14.1 T. This increase results from the relaxation interference between the C-13 chemical shielding anisotropy (CSA) and the H-1-C-13 dipolar interaction, as well as the simultaneous detection of H-1 and C-13 steady-state polarizations. Simulations indicate that the enhancement effect is maximal at currently available magnetic field strengths (less than or equal to 18.8 T). It is further shown that the recording of a pair of complementary TROSY experiments, denoted H-(alpha)- and H-(beta)-TROSY allows the accurate measurement of H-1-C-13 one-bond coupling constants from the cross-peak positions in the two spectra. An extension of the TROSY sequence is presented for recording high resolution 3D C-13-edited NOESY spectra.