Mercury(II) Site-Selective Binding to a DNA Hairpin. Relationship of Sequence-Dependent Intra- and Interstrand Cross-Linking to the Hairpin−Duplex Conformational Transition

Hg(II) interacted site selectively with only one of three deoxyribooligonucleotides examined; these "oligos" each had a different number of unmatched T residues. Thus, Hg(II) formed an intrastrand T-Hg-T cross-link between the first and fourth T residues of the hairpin, d(GCGCTTTTGCGC) (T4). The DNA strand formed a loop around the Hg, as if the Hg atom had been lassoed. The interactions of Hg(II) with two other oligos, d(ATGGGTTCCCAT) (T2) and d(GCGCTTTGCGC) (T3), were less specific. Previously, we found that at high DNA and salt concentrations, T2 was a mixture of hairpin and duplex forms while T3 and T4 had the hairpin form; modeling studies showed that in the free T4 hairpin the two T's at the ends of the (T)(4) loop form a T.T wobble base pair. Only in T4 are the T residues positioned to form an intrastrand cross-link readily. The Hg(II)-oligo adducts formed as a function of added Hg(II) were investigated by titrations monitored by UV, CD, and (1)H NMR spectroscopy. The appearance of a new set of (1)H signals with the concomitant decay of the free oligo (1)H signals indicated that 1:1 Hg(II):T2, 1.5:1 Hg(II):T3, and 1:1 Hg(II):T4 adducts were formed with Hg(NO(3))(2). In H(2)O, these adducts all had spectra with very downfield signals for the exchangeable TN(3)H and GN(1)H groups, a characteristic of base-paired regions. All upfield N(3)H signals from the (T)(2) and (T)(3) sequences of the free oligo disappeared in the spectra of the 1:1 Hg(II):T2 and 1.5:1 Hg(II):T3 adducts. The disappearance of the NH signals, the UV spectral changes, and the stoichiometries (1:1 Hg(II):T2 and 1.5:1 Hg(II):T3) indicate that these adducts are duplexes containing two and three T-Hg-T interstrand cross-links for T2 and T3, respectively. The (1)H and (13)C signals of the 1:1 Hg(II):T4 adduct in D(2)O were nearly completely assigned by 2D NMR spectroscopy. The spectrum of the adduct in H(2)O had only two of the four original TN(3)H signals from the (T)(4) sequence present in the spectrum of T4; this result is consistent with the presence of a TN3-Hg-TN3 cross-link. The (13)C chemical shift changes upon Hg(II) binding indicated that the TN3-Hg-TN3 cross-link was between the T's at each end of the (T)(4) loop. The NOESY, CD, and UV spectra were all consistent with a hairpin conformation for the 1:1 Hg(II):T4 adduct. A hairpin conformation also appeared reasonable from molecular modeling calculations. In conclusion, the length of the central (T)(n)() sequence influenced the type of T-Hg-T cross-link formed and, in turn, the conformation of the adducts. For (T)(2) and (T)(3), interstrand T-Hg-T cross-linking favored the duplex form. In contrast, for (T)(4), intrastrand T-Hg-T cross-linking stabilized the hairpin form.