title: Site-specific incorporation of N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) into oligonucleotides using modified 'ultra-mild' DNA synthesis.
abstract: Aromatic amino and nitro compounds are potent carcinogens found in the environment that exert their toxic effects by reacting with DNA following metabolic activation. One important adduct is N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF), which has been extensively used in studies of the mechanisms of DNA repair and mutagenesis. Despite the importance of dG-AAF adducts in DNA, an efficient method for its incorporation into DNA using solid-phase synthesis is still missing. We report the development of a modified 'ultra-mild' DNA synthesis protocol that allows the incorporation of dG-AAF into oligonucleotides of any length accessible by solid-phase DNA synthesis with high efficiency and independent of sequence context. Key to this endeavor was the development of improved deprotection conditions (10% diisopropylamine in methanol supplemented with 0.25 M of beta-mercaptoethanol) designed to remove protecting groups of commercially available 'ultra-mild' phosphoramidite building blocks without compromising the integrity of the exquisitely base-labile acetyl group at N8 of dG-AAF. We demonstrate the suitability of these oligonucleotides in the nucleotide excision repair reaction. Our synthetic approach should facilitate comprehensive studies of the mechanisms of repair and mutagenesis induced by dG-AAF adducts in DNA and should be of general use for the incorporation of base-labile functionalities into DNA.
authors.affiliation: Institute for Molecular Cancer Research, University of Zürich August Forel Strasse 7, 8008 Zürich, Switzerland.
authors.forename: Ludovic C J
authors.initials: LC
authors.lastname: Gillet
authors.forename: Jawad
authors.initials: J
authors.lastname: Alzeer
authors.forename: Orlando D
authors.initials: OD
authors.lastname: Schärer
mesh_terms: D015073:2-Acetylaminofluorene / Q000031:analogs & derivatives* / Q000737:chemistry*; D001671:Biochemistry / Q000379:methods; D018736:DNA Adducts / Q000138:chemical synthesis* / Q000737:chemistry; D004260:DNA Repair; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000737:chemistry*; D005449:Fluorenes / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis* / Q000737:chemistry
chemical_list: D018736:DNA Adducts; D005449:Fluorenes; D009838:Oligodeoxyribonucleotides; C031838:N-(deoxyguanosin-8-yl)-2-aminofluorene; C018776:N-(deoxyguanosin-8-yl)acetylaminofluorene; D015073:2-Acetylaminofluorene; D003849:Deoxyguanosine