Site-specific synthesis of aflatoxin B-1 adducts within an oligodeoxyribonucleotide containing the human p53 codon 249 sequence

This work describes the preparation of the cationic trans-8,9-dihydro-8-(N7 -guanyl)-9-hydroxyaflatoxin B-1 ((AFB)G) adducts at the positions correspon ding to G(746) or G(747), within the oligodeoxyribonucleotide d(GGAGGCCT) c ontaining the codon 249 sequence (underlined) of the p53 gene, using DNA tr iplexes to target adduction at the desired site. This approach enabled the successful preparation and purification of sufficient quantities of d(GGAG( AFB)GCCT) for NMR structural studies, using only standard phosphoramidites. The presence of multiple guanines in this oligodeoxynucleotide precluded t he direct reaction of d(GGAGGCCT); d(AGGCCTCC) with aflatoxin epoxide as a method for producing large quantities of site-specific adducts for physical studies. Of the multiple potential alkylation sites at guanine N7 in d(GGA GGCCT) d(AGGCCTCC), it was found that sites G(2) and G(5) exhibited approxi mately equal reactivity with aflatoxin B-1-exo-8,9-epoxide; the reactivity at site G(4) was reduced by approximately a factor of 2 as compared to that at G(2) or G(5). TO successfully prepare the sitespecific adducts, the p53 oligodeoxyribonucleotide was annealed with either the blocking strand d(CT CCATTTTCCT) or d(CCTCCATTTTCCTC) to form the corresponding partial triplexe s which targeted AFB(1) adduction either to G(4) or to G(5). Piperidine cle avage, followed by heating, confirmed that in each instance, the product co rresponded to the lone guanine not protected from adduction by the partial DNA tripler. The adducted oligodeoxyribonucleotides were examined with rega rd to purity by capillary electrophoresis. The primary advantage of this mo dified triple helix methodology is that it requires only standard phosphora midites; thus, it is applicable to large-scale preparations that are necess ary for NMR structural studies or other physical measurements.