Site-specific incorporation of N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) into oligonucleotides using modified 'ultra-mild' DNA synthesis

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 DN

Site-specific incorporation of N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) into oligonucleotides using modified 'ultra-mild' DNA synthesis

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

Application of fuzzy mathematical morphology for unsupervised color pixels classification

International audienc

Quantitative variability of 342 plasma proteins in a human twin population

The degree and the origins of quantitative variability of most human plasma proteins are largely unknown. Because the twin study design provides a natural opportunity to estimate the relative contribution of heritability and environment to different traits in human population, we applied here the highly accurate and reproducible SWATH mass spectrometry technique to quantify 1,904 peptides defining 342 unique plasma proteins in 232 plasma samples collected longitudinally from pairs of monozygotic and dizygotic twins at intervals of 2-7 years, and proportioned the observed total quantitative variability to its root causes, genes, and environmental and longitudinal factors. The data indicate that different proteins show vastly different patterns of abundance variability among humans and that genetic control and longitudinal variation affect protein levels and biological processes to different degrees. The data further strongly suggest that the plasma concentrations of clinical biomarkers need to be calibrated against genetic and temporal factors. Moreover, we identified 13 cis-SNPs significantly influencing the level of specific plasma proteins. These results therefore have immediate implications for the effective design of blood-based biomarker studies

Preparation of C8-amine and acetylamine adducts of 2'-deoxyguanosine suitably protected for DNA synthesis

[reaction: see text] C8-Amine and acetylamine adducts of 2'-deoxyguanosine were synthesized. Our approach provides solutions for the coupling of aromatic amines to a protected 8-bromo-2'-deoxyguanosine derivative, for the selective acetylation of the coupled adduct at N(8) and for a protecting group scheme preserving the integrity of the base-labile N(8) acetyl group during DNA synthesis