Recruitment of divalent metal ions by incorporation of 4-thio-2deoxythymidine or 4-thio-2-deoxyuridine into DNA

The modified nucleosides 4-thio-2-deoxyuridine (s4dU) and 4-thio-2-deoxythymidine (s^(4)dT) are incorporated into dinucleosides, and s4dT is incorporated into a DNA hairpin loop to provide divalent metal ion binding sites. Binding of two different metal ions to these sites is studied, including Cd(II) as an NMR spectroscopy probe and Cu(II) as a reactive metal ion for DNA cleavage. Binding of Cd(II) to 4-thiouridine (s^(4)U) and s^(4)dT nucleosides, s^(4)dU- and s^(4)dT-containing dinucleosides, and a hairpin loop oligonucleotide containing s^(4)dT is monitored by following the change in UV-vis absorbance of the thionucleosides at 340 nm and 21 °C in solutions containing 20.0–40 mM buffer, 1.00 M NaCl, and 15.0 mM BaCl2. Cd(II) binds to the N(3) deprotonated form of s^(4)dT with a binding constant (K=1.1×104 M^1) that is similar to that for Cd(II) binding to d(Tps^(4)T) (K=9.2×10^(3) M^(1)). Apparent binding constants (Kapp) at pH 7.7 of Cd(II) to dinucleosides d(Gps^(4)T), d(s^(4)TpG), and d(Gps^(4)U) are similar to those of their respective nucleosides s4U and s4dT, suggesting that neither the phosphate diester nor the second nucleoside has a major effect on Cd(II) binding. Binding of Cd(II) to s^(4)U and d(Gps^(4)U) is studied by use of 113Cd NMR and 1H NMR spectroscopy, respectively. Binding strength and stoichiometry of the Cd(II) complex with d(Gps^(4)U) as studied by 1H NMR spectroscopy are similar to that obtained by UV-vis spectroscopy. Cd(II) binds strongly to s^(4)dT in the loop portion of a DNA hairpin loop (Kapp=2.7×10^(3) M^(1) at pH 7.7). However, the hairpin loop is moderately destabilized by Cd(II) binding, with a decrease in Tm of 14 °C in the presence of 10.0 mM Cd(II) as determined by optical melting experiments. Cu(II) oxidizes s4dT to form the disulfide of s^(4)dT, limiting the usefulness of further studies with Cu(II).