A NMR strategy to unambiguously distinguish nucleic acid hairpin and duplex conformations applied to a Xist RNA A-repeat.
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.
Duszczyk
Malgorzata M
MM
Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
Zanier
Katia
K
Sattler
Michael
M
D001483:Base Sequence; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D009690:Nucleic Acid Conformation; D012330:RNA, Double-Stranded / Q000737:chemistry*; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated / Q000737:chemistry*; D012091:Repetitive Sequences, Nucleic Acid
D012330:RNA, Double-Stranded; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; C077885:XIST non-coding RNA