Sequence effects of aminofluorene-modified DNA duplexes: thermodynamic and circular dichroism properties

Abstract

Circular dichroism (CD) and UV-melting experiments were conducted with 16 oligodeoxynucleotides modified by the carcinogen 2-aminofluorene, whose sequence around the lesion was varied systematically [d(CTTCTNG[AF]NCCTC), N = G, A, C, T], to gain insight into the factors that determine the equilibrium between base-displaced stacked (S) and external B-type (B) duplex conformers. Differing stabilities among the duplexes can be attributed to different populations of S and B conformers. The AF modification always resulted in sequence-dependent thermal (T(m)) and thermodynamic (-DeltaG degrees ) destabilization. The population of B-type conformers derived from eight selected duplexes (i.e. -AG*N- and -CG*N-) was inversely proportional to the -DeltaG degrees and T(m) values, which highlights the importance of carcinogen/base stacking in duplex stabilization even in the face of disrupted Watson-Crick base pairing in S-conformation. CD studies showed that the extent of the adduct-induced negative ellipticities in the 290-350 nm range is correlated linearly with -DeltaG degrees and T(m), but inversely with the population of B-type conformations. Taken together, these results revealed a unique interplay between the extent of carcinogenic interaction with neighboring base pairs and the thermodynamic properties of the AF-modified duplexes. The sequence-dependent S/B heterogeneities have important implications in understanding how arylamine-DNA adducts are recognized in nucleotide excision repair.

Figure 1

(A) Chemical structures of AF-adduct (N-[deoxyguanosin-8-yl]-2-aminofluorene) and AAF-adduct (N-[deoxyguanosin-8-yl]-2-acetylaminofluorene). (B) Two prototype AF-induced conformations: an external binding B-type (B) conformer and a base-displaced stacked (S) conformer. The hydrophobic AF moiety is shown in CPK. The modified dG in the B-conformer maintains a Watson–Crick base pairing with the AF moiety protruding in the major groove, whereas the S-conformer adopts a base-displacement structure with the AF ring inserted into the helix. The duplex structures of sequence 5 were constructed with the literature protocols (16) and shown here for illustration purpose only.

Figure 2

CD spectral overlays of sequence 5 at 15°C; single-stranded AF-modified sequence (dotted line), unmodified duplex (dashed line), and AF-modified duplex (solid line).

Figure 3

CD spectral overlays of AF-modified duplexes in the 290–365 nm region at 15°C for (A) ‘S-curved’ and (B) ‘bucket-shaped’ sequences. (C) Calculation of CD_−/+ (Results).

Figure 4

Correlations of % B-conformation with (A) −ΔG°, (B) T_m and (C) CD_−/+ for eight selected AF-modified duplexes (sequences 5–12).