An intercalation inhibitor altering the target selectivity of DNA damaging agents: Synthesis of site-specific aflatoxin B(1) adducts in a p53 mutational hotspot

Aflatoxin B(1) (AFB(1)) is a potent human carcinogen implicated in the etiology of hepatocellular carcinoma. Upon metabolic activation to the reactive epoxide, AFB(1) forms DNA adducts primarily at the N7 position of guanines. To elucidate more fully the molecular mechanism of AFB(1)-induced mutagenesis, an intercalation inhibitor was designed to probe the effects of intercalation by AFB(1) epoxide on its reaction with DNA. DNA duplexes were prepared consisting of a target strand containing multiple potentially reactive guanines and a nontarget strand containing a cis-syn thymidine-benzofuran photoproduct. Because the covalently linked benzofuran moiety physically occupies an intercalation site, we reasoned that such a site would be rendered inaccessible to AFB(1) epoxide. By strategic positioning of this intercalation inhibitor in the intercalation site 5′ to a specific guanine, the adduct yield at that site was greatly diminished, indicating that intercalation by AFB(1) epoxide contributes favorably to adduct formation. Using this approach it has been possible to simplify the production of site-specifically modified oligonucleotides containing AFB(1) adducts in the sequence context of a p53 mutational hotspot. Moreover, we report herein isolation of site-specifically AFB(1)-modified oligonucleotides in sequences containing multiple guanines. Use of intercalation inhibitors will facilitate both investigation of the ability of other carcinogens to intercalate into DNA and the synthesis of specific carcinogen-DNA adducts.