A Silver-Specific DNAzyme with a New Silver Aptamer and Salt-Promoted Activity.

Most RNA-cleaving DNAzymes require a metal ion to interact with the scissile phosphate for activity. Therefore, few unmodified DNAzymes work with thiophilic metals because of their low affinity for phosphate. Recently, an Ag-specific Ag10c DNAzyme was reported via in vitro selection. Herein, Ag10c is characterized to rationalize the role of the strongly thiophilic Ag. Systematic mutation studies indicate that Ag10c is a highly conserved DNAzyme and its Ag binding is unrelated to C-Ag-C interaction. Its activity is enhanced by increasing Na concentrations in buffer. At the same metal concentration, activity decreases in the following order: Li > Na > K. Ag10c binds one Na ion and two Ag ions for catalysis. The pH-rate profile has a slope of ∼1, indicating a single deprotonation step. Phosphorothioate substitution at the scissile phosphate suggests that Na interacts with the pro-R oxygen of the phosphate, and dimethyl sulfate footprinting indicates that the DNAzyme loop is a silver aptamer binding two Ag ions. Therefore, Ag exerts its function allosterically, while the scissile phosphate interacts with Na, Li, Na, or Mg. This work suggests the possibility of isolating thiophilic metal aptamers based on DNAzyme selection, and it also demonstrates a new Ag aptamer.