Improved Sensitivity and Resolution in ¹H−¹³C NMR Experiments of RNA

Abstract

NMR studies of the structure and dynamics in RNA are greatly facilitated by the use of 13C-labeled molecules and 1H−13C correlation experiments. We demonstrate here that spin-state selective 13C frequency editing yields greatly improved sensitivity and resolution in 1H−13C 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% 13C-labeled 33-mer RNA at a magnetic field strength of 14.1 T. This increase results from the relaxation interference between the 13C chemical shielding anisotropy (CSA) and the 1H−13C dipolar interaction, as well as the simultaneous detection of 1H and 13C 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 1H−13C 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 13C-edited NOESY spectra.

Resolution-optimized NMR measurement of 1DCH, 1DCC and 2DCH residual dipolar couplings in nucleic acid bases

“…Here, we focus on the types of couplings that provide the highest normalized accuracy. Optimal results are obtained with TROSY-based pulse schemes (Brutscher et al, 1998;Pervushin et al, 1998;Fiala et al, 2000). Not only do these methods yield RDCs which provide excellent cross-validation (rms errors less than 3% of their potential range), they also yield very narrow bands for the isotropic values of the various J couplings.…”

The 3D NOESY-[ 1 H, 15 N, 1 H]-ZQ-TROSY NMR experiment with diagonal peak suppression

“…Secondly, compared to previous TROSY implementations in 3D heteronuclear-resolved NOE experiments (7,8), where transverse relaxation was optimized during the evolution of the heteronuclear spin and the directly observed 1 H dimension, the experiment of Fig. 1b affords optimization in all three dimensions and thus yields narrower lines and concomitantly increased peak heights in both dimensions of the all-important (Fig.…”

“…In these NOE experiments, the conventional [ 13 H]-heteronuclear singlequantum coherence or heteronuclear multiple-quantum coherence schemes (9) were substituted by the corresponding single-quantum TROSY-type chemical shift correlation schemes (2,4), whereby the desired transverse relaxation optimization was achieved along the 13 C frequency axis (7) or in the 15 …”

Structure determination and dynamics of protein–RNA complexes by NMR spectroscopy

“…This experiment is called IPAP for in-phase/anti-phase. Furthermore, if the faster relaxing TROSY upfield component is too broad to be accurately measured, the coupling constants can be determined by measuring the frequency difference in Hz between a resonance in a 15 presented, where 13 C-13 C homonuclear couplings were removed by using a fractionally 15% 13 C labeled 33-mer RNA [258].…”