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

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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 104 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

All Science Journal Classification (ASJC) codes

  • Catalysis
  • Organic Chemistry

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    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, 17(19), 5387-5392. https://doi.org/10.1002/chem.201003744