Quantitative 2D and 3D Γ-HCP Experiments for the Determination of the Angles α and ζ in the Phosphodiester Backbone of Oligonucleotides

摘要

The quantitative Γ-(HCP) experiment, a novel heteronuclear NMR pulse sequence for the determination of the RNA backbone angles α(O3′_i−1−P_i−O5′_i−C5′_i) and ζ(C3′_i−O3′_i−P_i+1−O5′_i+1) in 13C-labeled RNA, is introduced. The experiment relies on the interaction between the CH bond vector dipole and the 31P chemical shift anisotropy (CSA), which affects the relaxation of the 13C,31P double- and zero-quantum coherence and thus the intensity of the detectable magnetization. With the new pulse sequence, five different cross-correlated relaxation rates along the phosphodiester backbone can be measured in a quantitative manner, allowing projection-angle and torsion-angle restraints for the two backbone angles α and ζ to be extracted. Two versions of the pulse sequence optimized for the CH and CH₂ groups are introduced and demonstrated for a 14-mer cUUCGg tetraloop RNA model system and for a 27-mer RNA with a previously unknown structure. The restraints were incorporated into the calculation of a very high resolution structure of the RNA model system (Nozinovic, S.; et al. Nucleic Acids Res. 2010, 38, 683). Comparison with the X-ray structure of the cUUCGg tetraloop confirmed the high quality of the data, suggesting that the method can significantly improve the quality of RNA structure determination.