Figure 2.

Assays of the RadDz3 deoxyribozyme that cleaves ssDNA by a radical pathway. Incubation conditions: 70 mM HEPES, pH 7.5, combinations of 1 mM ZnCl₂, 20 mM MnCl₂, and 40 mM MgCl₂ as indicated, and 150 mM NaCl at 37 °C. (A) PAGE analysis of single-turnover DNA cleavage by RadDz3, using 5′-³²P-radiolabeled DNA substrate. Representative time points at t = 30 s, 5 h, and 24 h are shown. S = substrate; P = product. (B) Kinetic plots. For this data set, k_obs values (h⁻¹): Zn²⁺/Mn²⁺/Mg²⁺ 0.21, Zn²⁺/Mn²⁺ 0.22, Zn²⁺/Mg²⁺ 0.004, Mn²⁺/Mg²⁺ 0.17, Mn²⁺ 0.38. When Mn²⁺ was included at 1-300 µM along with 1 mM Zn²⁺ and 40 mM Mg²⁺, the DNA cleavage yield was the same as with Zn²⁺/Mg²⁺ in the absence of Mn²⁺ (data not shown), indicating that trace Mn²⁺ is not responsible for the Zn²⁺/Mg²⁺ reactivity. See the Experimental Section for quantitative analysis information on metal ion salts. (C) Determination of metal ion concentration dependence for Zn²⁺ (in the presence of 20 mM Mn²⁺) and Mn²⁺ alone. Data points with error bars were n = 2 (weighted averages; error bars by propagation from curve fit error); data points without error bars were n = 1. For Mn²⁺ alone, squares were fit, and triangles were not fit. Apparent K_d = 26 ± 2 mM, Hill coefficient n = 2.8 ± 0.4, k_max = 0.10 ± 0.01 h⁻¹.