Site-specific isotope labeling of long RNA for structural and mechanistic studies

Ikumi Kawahara, Kaichiro Haruta, Yuta Ashihara, Daichi Yamanaka, Mituhiro Kuriyama, Naoko Toki, Yoshinori Kondo, Kenta Teruya, Junya Ishikawa, Hiroyuki Furuta, Yoshiya Ikawa, Chojiro Kojima, Yoshiyuki Tanaka

    Research output: Contribution to journalArticlepeer-review

    8 Citations (Scopus)

    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 15N 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.

    Original languageEnglish
    Pages (from-to)e7
    JournalNucleic acids research
    Volume40
    Issue number1
    DOIs
    Publication statusPublished - Jan 2012

    All Science Journal Classification (ASJC) codes

    • Genetics

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