A viral genome containing an unstable aflatoxin B-1-N7-guanine DNA adduct situated at a unique site

A problem that has hindered the study of the biological properties of certain DNA adducts, such as those that form at the N7 atoms of purines, is their extreme chemical lability, Conditions are described for the construction of a single-stranded genome containing the chemically and thermally labile 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B-1 (AFB(1)-N7-Gua) adduct, the major DNA adduct of the potent liver carcinogen aflatoxin B-1 (AFB(1)). A 13mer oligonucleotide, d(CCTCTTCGAACTC), was allowed to react with the exo-8,9-epoxide of AFB(1) to form an oligonucleotide containing a single AFB(1)-N7-Gua (at the underlined guanine), This modified 13mer was 5'-phosphorylated and ligated into a gap in an M13 bacteriophage genome generated by annealing a 53mer uracil-containing scaffold to M13mp7L2 linearized by EcoRI, Following ligation, the scaffold was enzymatically removed with uracil DNA glycosylase and exonuclease III, The entire genome construction was complete within 3 h and was carried out at 16 degrees C, pH 6.6, conditions determined to be optimal for AFB(1)-N7-Gua stability, Characterization procedures indicated that the AFB(1)-N7-Gua genome was similar to 95% pure with a small (5%) contamination by unmodified genome, This construction scheme should be applicable to other chemically or thermally unstable DNA adducts.