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 ( Pfu AP) 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.