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 N6-(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.

Mutagenic Spectra Arising from Replication Bypass of the 2,6-Diamino-4-hydroxy-N5-methyl Formamidopyrimidine Adduct in Primate Cells

“…32 Furthermore, at least one polymerase of the B family was required to replicate past abasic sites in mammalian cells and this bypass generally proceeded via incorporation of dA opposite the lesion. 33 The MeFapy-dG lesion exists partially in the unnatural α-anomeric configuration, 10,34,35 and it is possible that this is recognized by the polymerases as an abasic site. Insertion of dA opposite the lesion site was found in bypass products generated by pol η or pol κ in the 5'-TXT-3' template, but not the 5'-TXG-3' template.…”

The Formamidopyrimidines: Purine Lesions Formed in Competition With 8-Oxopurines From Oxidative Stress

“…27 Both anomers of Fapy•dG reduced the UV-melting T m of the duplexes relative to an otherwise identical one containing a dG-dC base pair. However, α-Fapy•dG was considerably more destabilizing (ΔΔG = −5.3 kcal/mol) than β-Fapy•dG (ΔΔG = −1.5 kcal/mol).…”

Solution Structure of Duplex DNA Containing a β-Carba-Fapy-dG Lesion

“…We have recently reported a new synthesis approach for the incorporation of Fapy-dG in any sequence context, opening the door to future structural studies of lesion containing duplexes. 32 However, the high conformational and structural variability of Fapy-dG forecasts the extreme complexity of this ongoing endeavor. In contrast to normal nucleosides, the glycosidic bond of Fapy rearranges upon standing, producing an equilibrium mixture of α and β anomeric forms.…”