The use of an artificial nucleotide for polymerase-based recognition of carcinogenic O-6-alkylguanine DNA adducts

摘要

Enzymatic approaches for locating alkylation adducts at single-base resolution in DNA could enable new technologies for understanding carcinogenesis and supporting personalized chemotherapy. Artificial nucleotides that specifically pair with alkylated bases offer a possible strategy for recognition and amplification of adducted DNA, and adduct-templated incorporation of an artificial nucleotide has been demonstrated for a model DNA adduct O-6-benzylguanine by a DNA polymerase. In this study, DNA adducts of biological relevance, O-6-methylguanine (O-6-MeG) and O-6-carboxymethylguanine (O-6-CMG), were characterized to be effective templates for the incorporation of benzimidazole-derived 2'-deoxynucleoside-5'-O- triphosphates (BenziTP and BIMTP) by an engineered KlenTaq DNA polymerase. The enzyme catalyzed specific incorporation of the artificial nucleotide Benzi opposite adducts, with up to 150-fold higher catalytic efficiency for O-6-MeG over guanine in the template. Furthermore, addition of artificial nucleotide Benzi was required for full-length DNA synthesis during bypass of O-6-CMG. Selective incorporation of the artificial nucleotide opposite an O-6-alkylguanine DNA adduct was verified using a novel 2',3'-dideoxy derivative of BenziTP. The strategy was used to recognize adducts in the presence of excess unmodified DNA. The specific processing of BenziTP opposite biologically relevant O-6-alkylguanine adducts is characterized herein as a basis for potential future DNA adduct sequencing technologies.