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).