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.
- Brutscher B, Boisbouvier J, Pardi A, Marion D, Simorre JP (1998) Improved sensitivity and resolution in 1H-13C NMR experiments of RNA. J Am Chem Soc 120:11845–11851Google Scholar
- Fiala R, Jiang F, Sklenář V (1998) Sensitivity optimized HCN and HCNCH experiments for 13C/15N labeled oligonucleotides. J Biomol NMR 12:373–383Google Scholar
- Goddard TD, Kneller DG (2008) SPARKY 3: University of California, San Francisco, http://www.cgl.ucsf.edu/home/sparky
- Kay LE, Clore GM, Bax A, Gronenborn AM (1990) Four-dimensional heteronuclear triple-resonance NMR spectroscopy of interleukin-1-beta in solution. Science 249:411–414Google Scholar
- Marino JP, Schwalbe H, Anklin C, Bermel W, Crothers DM, Griesinger C (1994) Three-dimensional triple-resonance 1H, 13C, 31P experiment: sequential through-bond correlation of ribose protons and intervening phosphorus along the RNA oligonucleotide backbone. J Am Chem Soc 116:6472–6473CrossRefGoogle Scholar
- Marino JP, Diener JL, Moore PB, Griesinger C (1997) Multiple-quantum coherence dramatically enhances the sensitivity of CH and CH2 correlations in uniformly 13C-labeled RNA. J Am Chem Soc 119:7361–7366Google Scholar
- Riek R, Pervushin K, Fernandez C, Kainosho M, Wüthrich K (2001) [13C, 13C]- and [13C, 1H]-TROSY in a triple resonance experiment for ribose-base and intrabase correlations in nucleic acids. J Am Chem Soc 123:658–664Google Scholar
- Vuister GW, Clore GM, Gronenborn AM, Powers R, Garrett DS, Tschudin R, Bax A (1993) Increased resolution and improved spectral quality in four-dimensional 13C/13C-separated HMQC-NOESY-HMQC spectra using pulsed-field gradients. J Magn Reson Ser B 101:210–213Google Scholar