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 13 C-labeled molecules and 1 H− 13 C correlation experiments. We demonstrate here that spin-state selective 13 C frequency editing yields greatly improved sensitivity and resolution in 1 H− 13 C 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 √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% 13 C-labeled 33-mer RNA at a magnetic field strength of 14.1 T. This increase results from the relaxation interference between the 13 C chemical shielding anisotropy (CSA) and the 1 H− 13 C dipolar interaction, as well as the simultaneous detection of 1 H and 13 C steady-state polarizations. Simulations indicate that the enhancement effect is maximal at currently available magnetic field strengths (≤18.8 T). It is further shown that the recording of a pair of complementary TROSY experiments, denoted H ( α ) - and H ( β ) -TROSY allows the accurate measurement of 1 H− 13 C 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 13 C-edited NOESY spectra.