Automated and assisted RNA resonance assignment using NMR chemical shift statistics.
The three-dimensional structure determination of RNAs by NMR spectroscopy relies on chemical shift assignment, which still constitutes a bottleneck. In order to develop more efficient assignment strategies, we analysed relationships between sequence and (1)H and (13)C chemical shifts. Statistics of resonances from regularly Watson-Crick base-paired RNA revealed highly characteristic chemical shift clusters. We developed two approaches using these statistics for chemical shift assignment of double-stranded RNA (dsRNA): a manual approach that yields starting points for resonance assignment and simplifies decision trees and an automated approach based on the recently introduced automated resonance assignment algorithm FLYA. Both strategies require only unlabeled RNAs and three 2D spectra for assigning the H2/C2, H5/C5, H6/C6, H8/C8 and H1'/C1' chemical shifts. The manual approach proved to be efficient and robust when applied to the experimental data of RNAs with a size between 20 nt and 42 nt. The more advanced automated assignment approach was successfully applied to four stem-loop RNAs and a 42 nt siRNA, assigning 92-100% of the resonances from dsRNA regions correctly. This is the first automated approach for chemical shift assignment of non-exchangeable protons of RNA and their corresponding (13)C resonances, which provides an important step toward automated structure determination of RNAs.
Institute of Molecular Biology and Biophysics, ETH Zürich, 8093 Zürich, Switzerland, Institute of Biophysical Chemistry, Center for Biomolecular Magnetic Resonance, and Frankfurt Institute of Advanced Studies, 60438 Frankfurt am Main, Germany and Graduate School of Science and Engineering, Tokyo Metropolitan University, Hachioji, Tokyo 192-0397, Japan.
Thomas
T
Aeschbacher
Elena
E
Schmidt
Markus
M
Blatter
Christophe
C
Maris
Olivier
O
Duss
Frédéric H-T
FH
Allain
Peter
P
Güntert
Mario
M
Schubert
D000465:Algorithms; D003627:Data Interpretation, Statistical; D030561:Databases, Nucleic Acid; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D009690:Nucleic Acid Conformation; D012330:RNA, Double-Stranded / Q000737:chemistry*; D034741:RNA, Small Interfering / Q000737:chemistry; D012984:Software
D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering