The use of an artificial nucleotide for polymerase-based recognition of carcinogenic O⁶-alkylguanine DNA adducts

Abstract

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 O⁶-benzylguanine by a DNA polymerase. In this study, DNA adducts of biological relevance, O⁶-methylguanine (O⁶-MeG) and O⁶-carboxymethylguanine (O⁶-CMG), were characterized to be effective templates for the incorporation of benzimidazole-derived 2'-deoxynucleoside-5'-O-triphosphates ( BENZI: TP and BIM: TP) 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 O⁶-MeG over guanine in the template. Furthermore, addition of artificial nucleotide BENZI: was required for full-length DNA synthesis during bypass of O⁶-CMG. Selective incorporation of the artificial nucleotide opposite an O⁶-alkylguanine DNA adduct was verified using a novel 2',3'-dideoxy derivative of BENZI: TP. The strategy was used to recognize adducts in the presence of excess unmodified DNA. The specific processing of BENZI: TP opposite biologically relevant O⁶-alkylguanine adducts is characterized herein as a basis for potential future DNA adduct sequencing technologies.