title: Site-specific isotope labeling of long RNA for structural and mechanistic studies.
abstract: A site-specific isotope labeling technique of long RNA molecules was established. This technique is comprised of two simple enzymatic reactions, namely a guanosine transfer reaction of group I self-splicing introns and a ligation with T4 DNA ligase. The trans-acting group I self-splicing intron with its external cofactor, 'isotopically labeled guanosine 5'-monophosphate' (5'-GMP), steadily gave a 5'-residue-labeled RNA fragment. This key reaction, in combination with a ligation of 5'-remainder non-labeled sequence, allowed us to prepare a site-specifically labeled RNA molecule in a high yield, and its production was confirmed with (15)N NMR spectroscopy. Such a site-specifically labeled RNA molecule can be used to detect a molecular interaction and to probe chemical features of catalytically/structurally important residues with NMR spectroscopy and possibly Raman spectroscopy and mass spectrometry.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan.
authors.forename: Ikumi
authors.initials: I
authors.lastname: Kawahara
authors.forename: Kaichiro
authors.initials: K
authors.lastname: Haruta
authors.forename: Yuta
authors.initials: Y
authors.lastname: Ashihara
authors.forename: Daichi
authors.initials: D
authors.lastname: Yamanaka
authors.forename: Mituhiro
authors.initials: M
authors.lastname: Kuriyama
authors.forename: Naoko
authors.initials: N
authors.lastname: Toki
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Kondo
authors.forename: Kenta
authors.initials: K
authors.lastname: Teruya
authors.forename: Junya
authors.initials: J
authors.lastname: Ishikawa
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Furuta
authors.forename: Yoshiya
authors.initials: Y
authors.lastname: Ikawa
authors.forename: Chojiro
authors.initials: C
authors.lastname: Kojima
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Tanaka
mesh_terms: D011088:DNA Ligases; D007438:Introns; D007553:Isotope Labeling / Q000379:methods*; D019906:Nuclear Magnetic Resonance, Biomolecular; D012313:RNA / Q000737:chemistry*; D016337:RNA, Catalytic / Q000737:chemistry
chemical_list: D016337:RNA, Catalytic; C427204:hammerhead ribozyme; D012313:RNA; D011088:DNA Ligases
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