Book of Abstracts: Albany 2005
Probing the Conformational Heterogeneity of the Aminofluorene-modified DNA by 19F NMR Spectroscopy
Arylamines are an important group of mutagenic/carcinogenic carcinogens. Among the extensively studied arylamines are 2-aminofluorene (AF), N-2-Acetylaminofluorene (AAF), 4-aminobiphenyl (ABP), and aminopyrenes (APs), all of which produce N-deacetylated C8-substituted 2'-deoxyguanosine adducts as the major and the most persistent species. These lesions in DNA are known to exist in two major prototype conformations: a B-type (B) external conformer and a stacked (S) base-displaced conformer. It is well-established that the S/B conformational heterogeneity is sequence dependent and plays a key role in the mechanisms of arylamine mutagenesis. In the present study, we used 19F NMR spectroscopy to probe the sequence effect on the AF-induced S/B conformational heterogeneity of DNA duplexes. A series of fluorinated AF-modified 12-mer DNA duplexes were prepared as model probes. The sequences flanking the lesion (CTTCTNG*NCCTC: G*=dG-C8-FAF; N = dG, dC, dA, dT) are varied systematically. We conducted 2D chemical exchange and solvent-induced H/D isotope experiments for signal assignment. Temperature dependence spectra were also carried out for dynamic exchange NMR analysis. We discovered that: (i) the S/B conformational equilibrium exists for all the duplexes studied; (ii) the nature of flanking base pairs greatly influences the electronic environment (19F chemical shifts) of the inserted carcinogenic ring of S-conformers; (iii) the extent of S-conformer population is markedly sequence dependent (i.e., 3'-G > A > C > T); (iv) the S/B conformeric equilibrium follows line-shape changes characteristic of a two-site exchange process; thereby, repair and replication enzymes are likely to be confronted with the two conformations in vivo. These results provide valuable quantitative and dynamic insights into the sequence effects on the AF-induced S/B conformational heterogeneity and their muagenic consequences. The 19F NMR approach has shown to be as a powerful tool for investigating the conformational basis of adduct-initiated chemical carcinogenesis.
Supported by NIH R01CA98296.
Srinivasa Rao Meneni
Department of Biomedical and Pharmaceutical Sciences