The N7 imidazole nitrogen of guanine is the most susceptible site for DNA methylation, resulting in the formation of the cationic adduct 7-methylguanine (7MeG). Opening of the imidazole ring occurs upon exposure to hydroxide ion, producing the 2,6-diamino-4-hydroxy-N5-(methyl)-formamidopyrimidine (MeFapy-dG) lesion. This lesion is known to convert between α and β anomers, but the possibility of additional conformational isomers cannot be ruled out. We report progress towards characterizing this DNA adduct, including the synthesis and purification of a 12mer oligodeoxynucleotide in which the CH3 group of the MeFapy-dG is 13C-labeled in the sequence 5'-CATXATGACGCT-3'; X=13C-MeFapy-dG. The reverse phase HPLC chromatogram showed two equilibrating chromatographic peaks that were verified as the 13C-MeFapy-dG 12mer by MALDI mass spectrometry. It is hypothesized that the two equilibrating peaks are a result of anomerization of the MeFapy-dG adduct; such epimerization has been found to occur in other Fapy lesions. Investigation by NMR spectroscopy of the MeFapy-dG phosphoramidite has provided the first spectroscopic evidence that epimerization also occurs in the MeFapy-dG lesion. Eight unique spin systems of the deoxyribose ring were observed in a TOCSY experiment, indicative of eight isomers. NOE cross-peaks between the protons of the deoxyribose ring revealed that four of the species were the α anomer and four were the β anomer; the α anomer was the major component. Rotational isomers arising from rotation around the C5-N5 bond were observed in the β anomer. HSQC experiments of the 13C-labeled MeFapy-dG 12mer indicated the methyl group of MeFapy-dG is in ten and six unique chemical environments in single strand and in duplex, respectively. The methyl peaks were spilt into two distinct groups. A series of temperature dependent HSQC experiments of the single strand indicated that the two groups are caused by anomers rather than rotamers. In the NMR spectra of the duplex the C1H1'-C1H6, C1H1'-A2H8, A2H1'-A2H8, and A2H1'-T3H6 NOE cross-peaks exhibited doubling, as did corresponding NOE cross-peaks in the deoxyribose H2' and H2'' region of the spectrum. Tripling of the T3H1'-T3H6 and C21H5-C21H6 NOE cross-peaks and corresponding NOE cross-peaks for T3 in the deoxyribose H2' and H2'' region of the spectrum was also observed. These NMR data indicated the presence of multiple distinct chemical environments for nucleotides near the lesion.

Characterization of the 2,6-diamino-4-hydroxy-N5-(methyl)-formamidopyrimidine DNA lesion

254th National Meeting (2017)

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