title: Noncovalent spin labeling of riboswitch RNAs to obtain long-range structural NMR restraints.
abstract: 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.
authors.affiliation: Institute of Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance, Goethe University Frankfurt , Max-von-Laue-Strasse 7, 60438 Frankfurt, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Helmling
authors.forename: Irene
authors.initials: I
authors.lastname: Bessi
authors.forename: Anna
authors.initials: A
authors.lastname: Wacker
authors.forename: Kai A
authors.initials: KA
authors.lastname: Schnorr
authors.forename: Hendrik R A
authors.initials: HR
authors.lastname: Jonker
authors.forename: Christian
authors.initials: C
authors.lastname: Richter
authors.forename: Dominic
authors.initials: D
authors.lastname: Wagner
authors.forename: Michael
authors.initials: M
authors.lastname: Kreibich
authors.forename: Harald
authors.initials: H
authors.lastname: Schwalbe
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000235:genetics*; D020029:Base Pairing; D001483:Base Sequence; D004578:Electron Spin Resonance Spectroscopy; D045622:Entomoplasmataceae / Q000235:genetics* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial / Q000737:chemistry* / Q000235:genetics*; D058928:Riboswitch / Q000235:genetics*; D013113:Spin Labels
chemical_list: D052157:Aptamers, Nucleotide; D012329:RNA, Bacterial; D058928:Riboswitch; D013113:Spin Labels