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.

Figure 1

Commonly formed adducts of N-acetoxy-2-acetylaminofluorene with 2′-deoxyguanosine. N-AAAF can react with dG residues in DNA to form at least three different adducts: 3-(deoxyguanosin-N²-yl)-2-acetylaminofluorene (3) (dG-N²-AAF); N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (4) (dG-AAF); and N-(deoxyguanosin-8-yl)-2-aminofluorene (5) (dG-AF). dG, dG-N²-AAF and dG-AF are believed to have the base mainly in the syn conformation, while the anti conformation is favored in dG-AAF.

Figure 2

Site-specific incorporation of dG-AAF and dG-AF into oligonucleotides. Reaction conditions: (a) DMTr-Cl, pyridine (76%); (b) N-ethyldiisopropylamine, 2-cyanoethoxydiisopropylchloro-phosphoramidite, CH₂Cl₂ (86%); (c) modified ‘ultra-mild’ DNA synthesis as shown in Table 1; and (d) 1 M NaOH, 0.25 M β-mercaptoethanol.

Figure 3

Nucleoside composition analyses of 9-AAF (A) and 9-AF (B). The oligonucleotides 9-AAF and 9-AF were digested with snake venom phosphodiesterase and calf intestine phosphatase and the resulting nucleosides were separated using the HPLC gradient described in Materials and Methods. The elution times were as follows: dC = 3.4 min, dG = 5.5 min, T = 6.3 min, dA = 8.2 min, 9-AAF = 16.3 min, 9-AF = 16.7 min, dG-AAF = 24.2 min and dG-AF = 24.8 min.

Figure 4

NER dual incision activity on dG-AAF and dG-AF-containing plasmids. Plasmids containing site-specific dG-AAF or dG-AF residues were incubated with cell extracts prepared from HeLa cells. The 24mer to 32mer excision products containing dG-AAF or dG-AF were detected by annealing to a complementary oligonucleotide with a 5′-GpGpGpG overhang, which served as a template for end-labeling with [α-³²P]dCTP with sequenase. The reaction products were resolved on a 14% denaturing polyacrylamide gel. An MspI digest of pBR322 was used as a size marker and the position of the 27 and 35 nt bands are indicated.