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 β-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.

Conformational Differences of the C8-Deoxyguanosine Adduct of 2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) within the NarI Recognition Sequence

“…Our lab and others have utilized this reaction for the synthesis of C8-arylamine adducts of dGuo (33,36,(49)(50)(51)(52)(53)(54)(55)(56)(57)(58). The C8-dGuo adducts of IQ and AF were synthesized using this chemistry and incorporated into oligonucleotides via their phosphoramidite.…”

Multiple DNA Binding Domains Mediate the Function of the ERCC1-XPF Protein in Nucleotide Excision Repair

“…In Vitro NER Assay-Extracts derived from XPF-deficient XP2YO cells and plasmids containing dG-acetylaminofluorene (dG-AAF) or 1,3-intrastrand cisplatin (cis-Pt) lesions were prepared as described previously (45)(46)(47). For each reaction, 2 l of repair buffer (200 mM Hepes-KOH (pH 7.8), 25 mM MgCl 2 , 2.5 mM DTT, 10 mM ATP, 110 mM phosphocreatine (di-Tris salt, Sigma), and 1.8 mg/ml BSA), 0.2 l of creatine phosphokinase (2.5 mg/ml, sigma), 3 l of XPF-deficient cell extract (about 10 mg/ml), NaCl (to a final concentration of 70 mM), and different amounts of purified ERCC1-XPF in a total volume of 9 l were pre-warmed at 30°C for 10 min.…”

“…Quantification of the data revealed that the ERCC1 K247A/K281A also displayed the lowest binding affinity for Mutations in Multiple DNA Binding Domains Synergistically Affect NER Activity in Vitro-We then investigated the effect of these mutations on the overall NER reaction in the context of cell-free extracts. Plasmids containing site-specific bulky DNA substrates, either 1,3-intrastrand cisplatin crosslinks (cis-Pt) or acetylaminofluorene adducts of dG (dG-AAF), were incubated with XPF-deficient cell-free extract and wild-type or mutant ERCC1-XPF to observe the ability of the protein to excise DNA in the context of other NER proteins (45,46,52). XP-F extracts are devoid of ERCC1 and XPF and did not display any NER activity, but addition of wild-type ERCC1-XPF restored NER activity as shown previously (Fig.…”

Quantitative fluorescence nanospectroscopy of nucleotide excision repair : from single molecules to cells

“…N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (AAF) is one the important adducts which has been widely used in the studies of the mechanisms of DNA repair and mutagenesis [235]. AAF modified 44-mer oligonucleotide was a kind gift of JungEun Yeo and Orlando D. Schärer, Stony Brook University, NY, USA, and it was prepared as described in Ref.…”

“…AAF modified 44-mer oligonucleotide was a kind gift of JungEun Yeo and Orlando D. Schärer, Stony Brook University, NY, USA, and it was prepared as described in Ref. [235] and used for the preparation of Alexa488 dye labeled double-stranded 44 bps DNA substrate containing an AAF adduct (Alexa488- AAF-DNA in Fig. 6-1) by synthesizing with equimolar amount of its complementary strand.…”