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Journal/Book: J Am Chem Soc
Published: 2010
Pages: 10318-29
Volume: 132
Issue: 30
Accession no.: 185

Quantitative 2D and 3D Gamma-HCP Experiments for the Determination of the Angles alpha and zeta in the Phosphodiester Backbone of Oligonucleotides

Senada Nozinovic, Christian Richter, Jörg Rinnenthal, Boris Fürtig, Elke Duchardt-Ferner, Julia E. Weigand, Harald Schwalbe
The quantitative Γ-(HCP) experiment, a novel heteronuclear NMR pulse sequence for the determination of the RNA backbone angles alpha(O3′i-1-Pi-O5′i-C5′i) and zeta(C3′i-O3′i-Pi+1-O5′i+1) in 13Clabeled 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 zeroquantum 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 alpha and zeta 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.
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Prof. Dr. Harald Schwalbe
Institut für Organische Chemie und Chemische Biologie
Johann Wolfgang Goethe Universität
Max-von-Laue-Str. 7
D-60438 Frankfurt am Main
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