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

In 1994, the first DNAzyme named GR5 was reported, which specifically requires Pb²⁺ for its RNA cleavage activity. Three years later, the 8-17 DNAzyme was isolated. The 8-17 DNAzyme and the related 17E DNAzyme are also most active with Pb²⁺ , although other divalent metals can work as well. GR5 and 17E 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 17E might be a special form of GR5. To test this hypothesis, we performed systematic rational evolution experiments to gradually mutate GR5 toward 17E. By using the activity ratio in the presence of Pb²⁺ and Mg²⁺ for defining these two DNAzymes, the critical nucleotide was identified to be T₁₂ in 17E for metal specificity. In addition, G₉ in GR5 is a position not found in most 17E or 8-17 DNAzymes, and G₉ needs to be added to rescue GR5 activity if T₁₂ 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.