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: Mark Lukin, Conceição A S A Minetti, David P Remeta, Sivaprasad Attaluri, Francis Johnson, Kenneth J Breslauer, Carlos de Los Santos
• Journal: Nucleic acids research (Nucleic Acids Res) Vol. 39 Issue 13 Pg. 5776-89 (Jul 2011) ISSN: 1362-4962 [Electronic] England
• PMID: 21415012 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)

Chemical References

• DNA, Single-Stranded
• Formamides
• Furans
• Mutagens
• N6-(2-deoxy-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine
• Oligodeoxyribonucleotides
• Pyrimidines
• DNA

Topics

• Chromatography, Ion Exchange
• DNA (chemistry)
• DNA Damage
• DNA, Single-Stranded (chemistry)
• Formamides (chemistry)
• Furans (chemistry)
• Isomerism
• Mutagens (chemistry)
• Nucleic Acid Conformation
• Oligodeoxyribonucleotides (chemical synthesis, chemistry, isolation & purification)
• Pyrimidines (chemistry)
• Thermodynamics