4D Non-uniformly sampled C,C-NOESY experiment for sequential assignment of 13C,15N-labeled RNAs
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A 4D 13C(aromatic),13C(ribose)-edited NOESY experiment is introduced to improve sequential assignment of non-coding RNA, often hampered by a limited dispersion of 1H and 13C chemical shifts. The 13C-labeling of RNA is fully utilized by inclusion of two 13C evolution periods. These dimensions provide enhanced dispersion of resonances in the 4D spectrum. High spectral resolution is obtained using random non-uniform sampling in three indirect dimensions. The autocorrelation peaks are efficiently suppressed using band-selective pulses. Since the dynamic range of observed resonances is significantly decreased, the reconstruction of the 4D spectrum is greatly simplified. The experiment can replace two conventionally sampled 3D NOESY spectra (either ribose-13C- or aromatic-13C-separated), and remove most ambiguities encountered during sequential walks. The assignment strategy based on a homonuclear and 4D C,C-edited NOESY experiments is proposed and verified on a 34-nt RNA showing typical structure elements.
KeywordsMultidimensional NMR Non-uniform sampling Isotope labeled RNA Resonance assignment
This work was supported by Bio-NMR project funded by European Commission’s 7th Framework Program (contract No. 1618630) and the Slovenian Research Agency, the Ministry of Higher Education, Science and Technology of the Republic of Slovenia [P1-0242 and J1-4020]. J. S. thanks Polish National Science Centre for the financial support with the Grant No. 2012/05/N/ST4/01120. The study was carried out at the Biological and Chemical Research Centre, University of Warsaw, established within the project co-financed by European Union from the European Regional Development Fund under the Operational Programme Innovative Economy, 2007–2013.
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