Quantitative 2D and 3D Gamma-HCP experiments for the determination of the angles alpha and zeta in the phosphodiester backbone of oligonucleotides.
The quantitative Gamma-(HCP) experiment, a novel heteronuclear NMR pulse sequence for the determination of the RNA backbone angles alpha(O3'(i-1)-P(i)-O5'(i)-C5'(i)) and zeta(C3'(i)-O3'(i)-P(i+1)-O5'(i+1)) in (13)C-labeled RNA, is introduced. The experiment relies on the interaction between the CH bond vector dipole and the (31)P chemical shift anisotropy (CSA), which affects the relaxation of the (13)C,(31)P 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 alpha and zeta to be extracted. Two versions of the pulse sequence optimized for the CH and CH(2) 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.
Nozinovic
Senada
S
Institute for Organic Chemistry and Chemical Biology, Johann Wolfgang Goethe-University Frankfurt, Max-von-Laue-Strasse 7, 60438 Frankfurt am Main, Germany.
Richter
Christian
C
Rinnenthal
Jörg
J
Fürtig
Boris
B
Duchardt-Ferner
Elke
E
Weigand
Julia E
JE
Schwalbe
Harald
H
D001483:Base Sequence; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry*; D012313:RNA / Q000737:chemistry*
D009841:Oligonucleotides; D012313:RNA