Figure 8.

Assays of the RadDz6 deoxyribozyme that cleaves ssDNA. (A) Kinetic results from PAGE analysis of single-turnover DNA cleavage. Incubation conditions: 70 mM HEPES, pH 7.5, 1 mM ZnCl₂, 20 mM MnCl₂, 40 mM MgCl₂, 150 mM NaCl, and [H₂O₂] as indicated at 37 °C. (B) Kinetic results in the presence of KO₂. Incubation conditions as in panel A, with [KO₂] as indicated. (C) Kinetic results in the presence of various divalent metal ions. Incubation conditions as in panel A, with combinations of 1 mM ZnCl₂, 20 mM MnCl₂, and 40 mM MgCl₂ as indicated, each at 100 µM H₂O₂. For this data set, k_obs values (h⁻¹): Zn²⁺/Mn²⁺/Mg²⁺ 0.46, Zn²⁺/Mn²⁺ 0.50, Zn²⁺/Mg²⁺ 0.16, Mn²⁺/Mg²⁺ 0.35, Mn²⁺ 0.19. (D) MALDI mass spectrometry of DNA-catalyzed DNA cleavage products, formed with Zn²⁺/Mn²⁺/Mg²⁺ and 100 µM H₂O₂. The same DNA cleavage site as for RadDz3 is indicated by the data (cf. Figure 5). (E) Detection of the base propenal fragment released upon DNA cleavage using a colorimetric assay (cf. Figure 6). See the Experimental Section for assay details. The “product” observed for the three negative control experiments corresponds to background signal.