title: Transglutaminase-mediated synthesis of a DNA-(enzyme)n probe for highly sensitive DNA detection.
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.
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
authors.forename: Momoko
authors.initials: M
authors.lastname: Kitaoka
authors.forename: Yukito
authors.initials: Y
authors.lastname: Tsuruda
authors.forename: Yukari
authors.initials: Y
authors.lastname: Tanaka
authors.forename: Masahiro
authors.initials: M
authors.lastname: Goto
authors.forename: Masayuki
authors.initials: M
authors.lastname: Mitsumori
authors.forename: Kounosuke
authors.initials: K
authors.lastname: Hayashi
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Hiraishi
authors.forename: Katsuyuki
authors.initials: K
authors.lastname: Miyawaki
authors.forename: Sumihare
authors.initials: S
authors.lastname: Noji
authors.forename: Noriho
authors.initials: N
authors.lastname: Kamiya
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D002384:Catalysis; D004247:DNA / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D003856:Deoxyuracil Nucleotides / Q000737:chemistry; D004151:Dipeptides / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D005973:Glutamine / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D020027:Pyrococcus furiosus / Q000201:enzymology*; D011503:Transglutaminases / Q000378:metabolism*
chemical_list: D003856:Deoxyuracil Nucleotides; D004151:Dipeptides; D010455:Peptides; C432806:glutaminyl-glycine; D005973:Glutamine; D004247:DNA; D011503:Transglutaminases; D000469:Alkaline Phosphatase; D008239:Lysine