Probing Metal-Dependent Phosphate Binding for the Catalysis of the 17E DNAzyme

The RNA-cleaving 17E DNAzyme exhibits different levels of cleavage activity in the presence of various divalent metal ions, with Pb²⁺ giving the fastest cleavage. In this study, the metal-phosphate interaction is probed to understand the trend of activity with different metal ions. For the first-row transition metals, the lowest activity shown by Ni²⁺ correlates with the inhibition by the inorganic phosphate and its water ligand exchange rate, suggesting inner-sphere metal coordination. Cleavage activity with the two stereoisomers of the phosphorothioate-modified substrates, R_p and S_p, indicated that Mg²⁺, Mn²⁺, Fe²⁺, and Co²⁺ had the highest S_p:R_p activity ratio of >900. Comparatively, the activity was much less affected using the thiophilic metals, including Pb²⁺, suggesting inner-sphere coordination. The pH-rate profiles showed that Pb²⁺ was different than the rest of the metal ions in having a smaller slope and a similar fitted apparent pK_a and the pK_a of metal-bound water. Combining previous reports and our current results, we propose that Pb²⁺ most likely plays the role of a general acid while the other metal ions are Lewis acid catalysts interacting with the scissile phosphate.