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

The modified nucleosides 4-thio-2'-deoxyuridine (s4dU) and 4-thio-2'-deoxythymidine (s4dT) 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 (s4U) and s4dT nucleosides, s4dU- and s4dT-containing dinucleosides, and a hairpin loop oligonucleotide containing s4dT is monitored by following the change in UV-vis absorbance of the thionucleosides at 340 nm and 21 degrees C in solutions containing 20.0-40 mM buffer, 1.00 M NaCl, and 15.0 mM BaCl2. Cd(II) binds to the N3 deprotonated form of s4dT with a binding constant (K = 1.1 x 10(4) M(-1)) that is similar to that for Cd(II) binding to d(Tps4T) (K = 9.2 x 10(3) M(-1)). Apparent binding constants (Kapp) at pH 7.7 of Cd(II) to dinucleosides d(Gps4T), d(s4TpG), and d(Gps4U) 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 s4U and d(Gps4U) is studied by use of 113Cd NMR and 1H NMR spectroscopy, respectively. Binding strength and stoichiometry of the Cd(II) complex with d(Gps4U) as studied by 1H NMR spectroscopy are similar to that obtained by UV-vis spectroscopy. Cd(II) binds strongly to s4dT in the loop portion of a DNA hairpin loop (Kapp = 2.7 x 10(3) M(-1) at pH 7.7). However, the hairpin loop is moderately destabilized by Cd(II) binding, with a decrease in T(m) of 14 degrees 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 s4dT, limiting the usefulness of further studies with Cu(II).