Noncovalent Spin Labeling of Riboswitch RNAs To Obtain Long-Range Structural NMR Restraints
| Autor: | Anna Wacker, Christina Helmling, Hendrik R. A. Jonker, Dominic Wagner, Michael Kreibich, Harald Schwalbe, Christian Richter, Kai A. Schnorr, Irene Bessi |
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| Rok vydání: | 2014 |
| Předmět: |
Models
Molecular Riboswitch Magnetic Resonance Spectroscopy Molecular Sequence Data 010402 general chemistry 01 natural sciences Biochemistry 03 medical and health sciences Nuclear magnetic resonance Base Pairing 030304 developmental biology 0303 health sciences Base Sequence Chemistry Relaxation (NMR) Electron Spin Resonance Spectroscopy General Medicine Site-directed spin labeling Aptamers Nucleotide 0104 chemical sciences RNA Bacterial Crystallography Nucleic Acid Conformation Molecular Medicine Spin Labels Entomoplasmataceae |
| Zdroj: | ACS Chemical Biology |
| ISSN: | 1554-8937 1554-8929 |
| DOI: | 10.1021/cb500050t |
| Popis: | Paramagnetic relaxation enhancement (PRE) NMR is a powerful method to study structure, dynamics and function of proteins. Up to now, the application of PRE NMR on RNAs is a significant challenge due to the limited size of chemically synthesized RNA. Here, we present a noncovalent spin labeling strategy to spin label RNAs in high yields required for NMR studies. The approach requires the presence of a helix segment composed of about 10 nucleotides (nt) but is not restricted by the size of the RNA. We show successful application of this strategy on the 2'dG sensing aptamer domain of Mesoplasma florum (78 nt). The aptamer domain was prepared in two fragments. A larger fragment was obtained by biochemical means, while a short spin labeled fragment was prepared by chemical solid-phase synthesis. The two fragments were annealed noncovalently by hybridization. We performed NMR, cw-EPR experiments and gel shift assays to investigate the stability of the two-fragment complex. NMR analysis in (15)N-TROSY and (1)H,(1)H-NOESY spectra of both unmodified and spin labeled aptamer domain show that the fragmented system forms a stable hybridization product, is in structural agreement with the full length aptamer domain and maintains its function. Together with structure modeling, experimentally determined (1)H-Γ2 rates are in agreement with reported crystal structure data and show that distance restraints up to 25 Å can be obtained from NMR PRE data of RNA. |
| Databáze: | OpenAIRE |
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