The Two Classic Pb2+‐Selective DNAzymes Are Related: Rational Evolution for Understanding Metal Selectivity

Abstract In 1994, the first DNAzyme named GR5 was reported, which specifically requires Pb 2+ for its RNA cleavage activity. Three years later, the 8‐17 DNAzyme was isolated. The 8‐17 DNAzyme and the related 17 E DNAzyme are also most active with Pb 2+ , although other divalent metals can work as well. GR5 and 17 E have the same substrate sequence, and their catalytic loops in the enzyme strands also have a few similar and conserved nucleotides. Considering these, we hypothesized that 17 E might be a special form of GR5. To test this hypothesis, we performed systematic rational evolution experiments to gradually mutate GR5 toward 17 E . By using the activity ratio in the presence of Pb 2+ and Mg 2+ for defining these two DNAzymes, the critical nucleotide was identified to be T 12 in 17 E for metal specificity. In addition, G 9 in GR5 is a position not found in most 17 E or 8‐17 DNAzymes, and G 9 needs to be added to rescue GR5 activity if T 12 becomes a cytosine. This study highlights the links between these two classic and widely used DNAzymes, and offers new insight into the sequence–activity relationship related to metal selectivity.