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

Abstract

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_p 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 Mg²⁺. This work suggests the possibility of isolating thiophilic metal aptamers based on DNAzyme selection, and it also demonstrates a new Ag⁺ aptamer.