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

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).