Nucleic Acids Research Advance Access originally published online on November 5, 2008
Nucleic Acids Research 2008 36(22):7068-7077; doi:10.1093/nar/gkn776
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Nucleic Acids Research, 2008, Vol. 36, No. 22 7068-7077
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Structural Biology |
A NMR strategy to unambiguously distinguish nucleic acid hairpin and duplex conformations applied to a Xist RNA A-repeat
1Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, 2Institute of Structural Biology, Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764 Neuherberg and 3Munich Center for Integrated Protein Science and Chair Biomolecular NMR, Department Chemie, Technische Universität München, Lichtenbergstr. 4, 85747 Garching, Germany
*To whom correspondence should be addressed. Tel: +49 89 289 13418; Fax: +49 89 289 13869; Email: sattler{at}helmholtz-muenchen.de
Received August 21, 2008. Revised October 2, 2008. Accepted October 8, 2008.
All RNA sequences that fold into hairpins possess the intrinsic potential to form intermolecular duplexes because of their high self-complementarity. The thermodynamically more stable duplex conformation is favored under high salt conditions and at high RNA concentrations, posing a challenging problem for structural studies of small RNA hairpin conformations. We developed and applied a novel approach to unambiguously distinguish RNA hairpin and duplex conformations for the structural analysis of a Xist RNA A-repeat. Using a combination of a quantitative HNN-COSY experiment and an optimized double isotope-filtered NOESY experiment we could define the conformation of the 26-mer A-repeat RNA. In contrast to a previous secondary structure prediction of a double hairpin structure, the NMR data show that only the first predicted hairpin is formed, while the second predicted hairpin mediates dimerization of the A-repeat by duplex formation with a second A-repeat. The strategy employed here will be generally applicable to identify and quantify populations of hairpin and duplex conformations and to define RNA folding topology from inter- and intra-molecular base-pairing patterns.
Present address: Katia Zanier, Ecole Supérieure de Biotechnologie de Strasbourg, Institut Gilbert Laustriat (UMR 7175 LC1), Boulevard Sébastien Brant, BP 10413, 67412 Illkirch, France