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 Kd 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+.