Esterase 2-oligodeoxynucleotide conjugates as sensitive reporter for electrochemical detection of nucleic acid hybridization.
A thermostable, single polypeptide chain enzyme, esterase 2 from Alicyclobacillus acidocaldarius, was covalently conjugated in a site specific manner with an oligodeoxynucleotide. The conjugate served as a reporter enzyme for electrochemical detection of DNA hybridization. Capture oligodeoxynucleotides were assembled on gold electrode via thiol-gold interaction. The esterase 2-oligodeoxynucleotide conjugates were brought to electrode surface by DNA hybridization. The p-aminophenol formed by esterase 2 catalyzed hydrolysis of p-aminophenylbutyrate was amperometrically determined. Esterase 2 reporters allows to detect approximately 1.5 x 10(-18)mol oligodeoxynucleotides/0.6 mm2 electrode, or 3 pM oligodeoxynucleotide in a volume of 0.5 microL. Chemically targeted, single site covalent attachment of esterase 2 to an oligodeoxynucleotide significantly increases the selectivity of the mismatch detection as compared to widely used, rather unspecific, streptavidin/biotin conjugated proteins. Artificial single nucleotide mismatches in a 510-nucleotide ssDNA could be reliably determined using esterase 2-oligodeoxynucleotide conjugates as a reporter.
Wang
Yiran
Y
Laboratorium für Biochemie, Universität Bayreuth, Universitätsstr. 30, 95440 Bayreuth, Germany.
Stanzel
Manfred
M
Gumbrecht
Walter
W
Humenik
Martin
M
Sprinzl
Mathias
M
D020137:Base Pair Mismatch; D001710:Biotin; D004247:DNA / Q000032:analysis; D004563:Electrochemistry / Q000379:methods*; D004950:Esterases / Q000378:metabolism*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D012680:Sensitivity and Specificity
D009838:Oligodeoxyribonucleotides; D001710:Biotin; D004247:DNA; D004950:Esterases