Folding of the silver aptamer in a DNAzyme probed by 2-aminopurine fluorescence

The RNA-cleaving Ag10c DNAzyme was recently isolated via in vitro selection and it can bind two Ag⁺ ions for activity. The Ag10c contains a well-defined Ag⁺ binding aptamer as indicated by DMS footprinting. Since aptamer binding is often accompanied with conformational changes, we herein used 2-aminopurine (2AP) to probe its folding in the presence of Ag⁺. The Ag10c was respectively labeled with 2AP at three different positions, both in the substrate strand and in the enzyme strand, one at a time. Ag⁺-induced folding was observed at the substrate cleavage junction and the A9 position of the enzyme strand, consistent with aptamer binding. The measured K_d at the A9 position was 18 μM Ag⁺ with a Hill coefficient of 2.17, similar to those obtained from the previous cleavage activity based assays. However, labeling a 2AP at the A2 position inhibited the activity and folding. Compared to other metal ions, Ag⁺ has a unique sigmoidal folding profile indicative of multiple silver binding cooperatively. This suggests that Ag⁺ can induce a local folding in the enzyme loop and this folding is important for activity. This study provides important biophysical insights into this new DNAzyme, suggesting the possibility of designing folding-based biosensors for Ag⁺.