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

Abstract

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 °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 s⁴dT with a binding constant (K=1.1×10⁴ M⁻¹) that is similar to that for Cd(II) binding to d(Tps⁴T) (K=9.2×10³ M⁻¹). Apparent binding constants (K_app) 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 (K_app=2.7×10³ 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 °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).