title: Novel post-synthetic generation, isomeric resolution, and characterization of Fapy-dG within oligodeoxynucleotides: differential anomeric impacts on DNA duplex properties.
abstract: Accumulation of damaged guanine nucleobases within genomic DNA, including the imidazole ring opened N(6)-(2-Deoxy-α,β-D-erythro-pentafuranosyl)-2,6-diamino-4-hydroxy-5-formylamidopyrimidine (Fapy-dG), is associated with progression of age-related diseases and cancer. To evaluate the impact of this mutagenic lesion on DNA structure and energetics, we have developed a novel synthetic strategy to incorporate cognate Fapy-dG site-specifically within any oligodeoxynucleotide sequence. The scheme involves the synthesis of an oligonucleotide precursor containing a 5-nitropyrimidine moiety at the desired lesion site via standard solid-phase procedures. Following deprotection and isolation, the Fapy-dG lesion is generated by catalytic hydrogenation and subsequent formylation. NMR assignment of the Fapy-dG lesion (X) embedded within a TXT trimer reveals the presence of rotameric and anomeric species. The latter have been characterized by synthesizing the tridecamer oligodeoxynucleotide d(GCGTACXCATGCG) harboring Fapy-dG as the central residue and developing a protocol to resolve the isomeric components. Hybridization of the chromatographically isolated fractions with their complementary d(CGCATGCGTACGC) counterpart yields two Fapy-dG·C duplexes that are differentially destabilized relative to the canonical G·C parent. The resultant duplexes exhibit distinct thermal and thermodynamic profiles that are characteristic of α- and β-anomers, the former more destabilizing than the latter. These anomer-specific impacts are discussed in terms of differential repair enzyme recognition, processing and translesion synthesis.
authors.affiliation: Department of Pharmacological Sciences, School of Medicine, Stony Brook University, Stony Brook, NY 11794-8651, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Lukin
authors.forename: Conceição A S A
authors.initials: CA
authors.lastname: Minetti
authors.forename: David P
authors.initials: DP
authors.lastname: Remeta
authors.forename: Sivaprasad
authors.initials: S
authors.lastname: Attaluri
authors.forename: Francis
authors.initials: F
authors.lastname: Johnson
authors.forename: Kenneth J
authors.initials: KJ
authors.lastname: Breslauer
authors.forename: Carlos
authors.initials: C
authors.lastname: de Los Santos
mesh_terms: D002852:Chromatography, Ion Exchange; D004247:DNA / Q000737:chemistry; D004249:DNA Damage; D004277:DNA, Single-Stranded / Q000737:chemistry; D005559:Formamides / Q000737:chemistry*; D005663:Furans / Q000737:chemistry*; D007536:Isomerism; D009153:Mutagens / Q000737:chemistry; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification; D011743:Pyrimidines / Q000737:chemistry*; D013816:Thermodynamics
chemical_list: D004277:DNA, Single-Stranded; D005559:Formamides; D005663:Furans; D009153:Mutagens; C435881:N6-(2-deoxy-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine; D009838:Oligodeoxyribonucleotides; D011743:Pyrimidines; D004247:DNA