The formation of DNA adducts can initiate the process of mutagenesis. The lack of methods to determine low abundance DNA adducts within particular DNA sequences limits our understanding of the contributions of alkylation to mutagenesis. The use of artificial nucleotides incorporated opposite DNA adducts by engineered polymerases is a potential strategy for site-specific detection of DNA adducts. In previous studies, we created artificial nucleotides that were incorporated specifically opposite O⁶-alkyl-guanine (G) adducts by a Taq polymerase mutant.[1] In this work, we designed and synthetized a new artificial nucleotide with an extended ρ-surface base that enables stronger enzymatic interactions, while retaining high and selective hybridization opposite O⁶-alkylG. We made significant advances by combining the incorporation of the new artificial nucleotide opposite a biologically relevant O⁶-alkylG lesion, i.e. O⁶-carboxymethylG(CMG), with analytical readouts. We measured and characterized the enzymatic process, and we established a mass spectrometric method for quantifying the incorporated artificial nucleotide as a marker for the presence and sequence location of O⁶-CMG. The results of this work advance chemical and biochemical tools for linking DNA alkylation to mutagenesis and for evaluating DNA adducts as potential diagnostic biomarkers.

[1] Laura A. Wyss, Arman Nilforoushan, Fritz Eichenseher, Ursina Suter, Nina Blatter, Andreas Marx, Shana J. Sturla, J. Am. Chem. Soc., 2015, 137, 1, 30–33