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 journalArticle

7 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 1 2012

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Isotope Labeling
RNA
Guanosine Monophosphate
Introns
Ligation
Magnetic Resonance Spectroscopy
DNA Ligases
Raman Spectrum Analysis
Guanosine
Mass Spectrometry

All Science Journal Classification (ASJC) codes

  • Genetics

Cite this

Kawahara, I., Haruta, K., Ashihara, Y., Yamanaka, D., Kuriyama, M., Toki, N., ... Tanaka, Y. (2012). Site-specific isotope labeling of long RNA for structural and mechanistic studies. Nucleic acids research, 40(1), e7. https://doi.org/10.1093/nar/gkr951

Site-specific isotope labeling of long RNA for structural and mechanistic studies. / Kawahara, Ikumi; Haruta, Kaichiro; Ashihara, Yuta; Yamanaka, Daichi; Kuriyama, Mituhiro; Toki, Naoko; Kondo, Yoshinori; Teruya, Kenta; Ishikawa, Junya; Furuta, Hiroyuki; Ikawa, Yoshiya; Kojima, Chojiro; Tanaka, Yoshiyuki.

In: Nucleic acids research, Vol. 40, No. 1, 01.01.2012, p. e7.

Research output: Contribution to journalArticle

Kawahara, I, Haruta, K, Ashihara, Y, Yamanaka, D, Kuriyama, M, Toki, N, Kondo, Y, Teruya, K, Ishikawa, J, Furuta, H, Ikawa, Y, Kojima, C & Tanaka, Y 2012, 'Site-specific isotope labeling of long RNA for structural and mechanistic studies', Nucleic acids research, vol. 40, no. 1, pp. e7. https://doi.org/10.1093/nar/gkr951
Kawahara I, Haruta K, Ashihara Y, Yamanaka D, Kuriyama M, Toki N et al. Site-specific isotope labeling of long RNA for structural and mechanistic studies. Nucleic acids research. 2012 Jan 1;40(1):e7. https://doi.org/10.1093/nar/gkr951
Kawahara, Ikumi ; Haruta, Kaichiro ; Ashihara, Yuta ; Yamanaka, Daichi ; Kuriyama, Mituhiro ; Toki, Naoko ; Kondo, Yoshinori ; Teruya, Kenta ; Ishikawa, Junya ; Furuta, Hiroyuki ; Ikawa, Yoshiya ; Kojima, Chojiro ; Tanaka, Yoshiyuki. / Site-specific isotope labeling of long RNA for structural and mechanistic studies. In: Nucleic acids research. 2012 ; Vol. 40, No. 1. pp. e7.
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