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

Index: Chemistry 17 , 5387-5392, (2011)

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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.Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.