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 corresponding to G(746) or G(747), within the oligodeoxyribonucleotide d(GGAGGCCT) containing the codon 249 sequence (underlined) of the p53 gene, using DNA triplexes 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 the 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(GGAGGCCT). d(AGGCCTCC), it was found that sites G(2) and G(5) exhibited approximately 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 site-specific adducts, the p53 oligodeoxyribonucleotide was annealed with either the blocking strand d(CTCCATTTTCCT) or d(CCTCCATTTTCCTC) to form the corresponding partial triplexes which targeted AFB(1) adduction either to G(4) or to G(5). Piperidine cleavage, followed by heating, confirmed that in each instance, the product corresponded to the lone guanine not protected from adduction by the partial DNA triplex. The adducted oligodeoxyribonucleotides were examined with regard to purity by capillary electrophoresis. The primary advantage of this modified triple helix methodology is that it requires only standard phosphoramidites; thus, it is applicable to large-scale preparations that are necessary for NMR structural studies or other physical measurements.
Jones
W R
WR
Department of Chemistry and Center in Molecular Toxicology, Vanderbilt University, Nashville, Tennessee 37235, USA.
Johnston
D S
DS
Stone
M P
MP
D016604:Aflatoxin B1 / Q000737:chemistry*; D001345:Autoradiography; D002273:Carcinogens / Q000737:chemistry*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003062:Codon; D018736:DNA Adducts / Q000138:chemical synthesis*; D004591:Electrophoresis, Polyacrylamide Gel; D004852:Epoxy Compounds / Q000138:chemical synthesis / Q000737:chemistry; D016158:Genes, p53 / Q000235:genetics*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry* / Q000235:genetics; D013056:Spectrophotometry, Ultraviolet
D002273:Carcinogens; D003062:Codon; D018736:DNA Adducts; D004852:Epoxy Compounds; D009838:Oligodeoxyribonucleotides; D016604:Aflatoxin B1