An Efficient Procedure for Assignment of the Proton, Carbon and Nitrogen Resonances in 13C/ 15N Labeled Nucleic Acids

Abstract

An efficient method is presented for the assignment of the proton, carbon, and nitrogen resonances in the NMR spectra of isotopically labeled nucleic acids. The assignment strategy starts by identifying all protons and carbons belonging to the same sugar ring through application of a set of 2D or 3D heteronuclear HCCH NMR experiments. Next the individual sugar rings are connected to their corresponding bases through intra-residue 1 H- 1H nuclear Overhauser effects (NOEs) observed in a 3D ( 1H, 13C, 1H) NOESY-HMQC experiment. Sequential NOE connectivities observed in this experiment are then used to assign each residue in the nucleotide sequence. The imino protons and nitrogens, and the cytidine amino protons and nitrogens, are assigned by 2D ( 15N, 1H) HMQC and 3D ( 1H, 15N, 1 H) NOESY-HMQC experiments in H 2O. This assignment procedure is illustrated on the 99% 13C/ 15N labeled RNA duplex r(GGCGCUUGCGUC) 2. The application of these multi-dimensional heteronuclear magnetic resonance experiments enormously simplifies the resonance assignment of nucleic acids and allows assignment of many more protons, carbons and nitrogens than was possible using standard techniques on unlabeled molecules. Since a larger percentage of the protons can now be assigned by these experiments, much more NMR structural information can be obtained which will significantly extend the size limit for solution structure determinations of RNAs.