Transglutaminase-mediated synthesis of a DNA-(Enzyme) n probe for highly sensitive DNA detection

Momoko Kitaoka, Yukito Tsuruda, Yukari Tanaka, Masahiro Goto, Masayuki Mitsumori, Kounosuke Hayashi, Yoshiyuki Hiraishi, Katsuyuki Miyawaki, Sumihare Noji, Noriho Kamiya

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

A new synthetic strategy for DNA-enzyme conjugates with a novel architecture was explored using a natural cross-linking catalyst, microbial transglutaminase (MTG). A glutamine-donor substrate peptide of MTG was introduced at the 5-position on the pyrimidine of deoxyuridine triphosphate to prepare a DNA strand with multiple glutamine-donor sites by polymerase chain reaction (PCR). A substrate peptide that contained an MTG-reactive lysine residue was fused to the N terminus of a thermostable alkaline phoshatase from Pyrococcus furiosus (PfuAP) by genetic engineering. By combining enzymatically the substrate moieties of MTG introduced to the DNA template and the recombinant enzyme, a DNA-(enzyme) n conjugate with 1:n stoichiometry was successfully obtained. The enzyme/DNA ratio of the conjugate increased as the benzyloxycarbonyl-L-glutaminylglycine (Z-QG) moiety increased in the DNA template. The potential utility of the new conjugate decorated with signaling enzymes was validated in a dot blot hybridization assay. The DNA-(enzyme) n probe could clearly detect 10 4 copies of the target nucleic acid with the complementary sequence under harsh hybridization conditions, thereby enabling a simple detection procedure without cumbersome bound/free processes associated with a conventional hapten-antibody reaction-based DNA-detection system. DNA detector: A glutamine-modified DNA probe was synthesized by the polymerase chain reaction by using a glutamine-modified 2′-deoxyuridine 5′-triphosphate analogue as a substrate of DNA polymerase. The DNA-(alkaline phosphatase) n conjugate probe was highly sensitive and could directly visualize the target DNA bound on a membrane immediately after hybridization (see graphic).

Original languageEnglish
Pages (from-to)5387-5392
Number of pages6
JournalChemistry - A European Journal
Volume17
Issue number19
DOIs
Publication statusPublished - May 2 2011

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Transglutaminases
DNA
Enzymes
Glutamine
Polymerase chain reaction
Substrates
glutaminyl-glycine
Peptides
Genetic engineering
Haptens
DNA Probes
DNA-Directed DNA Polymerase
Stoichiometry
Nucleic acids
Phosphatases
Nucleic Acids
Lysine
Alkaline Phosphatase
Assays
Antibodies

All Science Journal Classification (ASJC) codes

  • Catalysis
  • Organic Chemistry

Cite this

Transglutaminase-mediated synthesis of a DNA-(Enzyme) n probe for highly sensitive DNA detection . / Kitaoka, Momoko; Tsuruda, Yukito; Tanaka, Yukari; Goto, Masahiro; Mitsumori, Masayuki; Hayashi, Kounosuke; Hiraishi, Yoshiyuki; Miyawaki, Katsuyuki; Noji, Sumihare; Kamiya, Noriho.

In: Chemistry - A European Journal, Vol. 17, No. 19, 02.05.2011, p. 5387-5392.

Research output: Contribution to journalArticle

Kitaoka, M, Tsuruda, Y, Tanaka, Y, Goto, M, Mitsumori, M, Hayashi, K, Hiraishi, Y, Miyawaki, K, Noji, S & Kamiya, N 2011, ' Transglutaminase-mediated synthesis of a DNA-(Enzyme) n probe for highly sensitive DNA detection ', Chemistry - A European Journal, vol. 17, no. 19, pp. 5387-5392. https://doi.org/10.1002/chem.201003744
Kitaoka, Momoko ; Tsuruda, Yukito ; Tanaka, Yukari ; Goto, Masahiro ; Mitsumori, Masayuki ; Hayashi, Kounosuke ; Hiraishi, Yoshiyuki ; Miyawaki, Katsuyuki ; Noji, Sumihare ; Kamiya, Noriho. / Transglutaminase-mediated synthesis of a DNA-(Enzyme) n probe for highly sensitive DNA detection In: Chemistry - A European Journal. 2011 ; Vol. 17, No. 19. pp. 5387-5392.
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