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

Abstract

In 1994, the first DNAzyme namedG R5 was reported,w hich specifically requires Pb 2 + for its RNA cleavage activity.T hree years later,the 8-17 DNAzyme was isolated. The 8-17 DNAzyme and the related 17E DNAzyme are also most active with Pb 2 + , although other divalent metals can work as well. GR5 and 17E have the same substrates equence, and their catalytic loopsi n the enzyme strandsa lso have af ew similar and conserved nucleotides. Considering these, we hypothesizedt hat 17E might be aspecial form of GR5. To test this hypothesis, we performed systematic rational evolution experiments to gradually mutate GR5 toward1 7 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 17E for metal specificity. In addition, G 9 in GR5i sap ositionn ot found in most 17E or 8-17 DNAzymes, and G 9 needs to be added to rescue GR5 activity if T 12 becomes ac ytosine. This study highlightst he links between these two classic and widely used DNAzymes, and offers new insight into the sequence-activity relationshipr elated to metal selectivity.DNAzymes are DNA-based catalysts. [1] With excellent stability, high catalytic efficiency,programmability,and ease of modification, DNAzymes have been explored for various applications such as metal sensing,a ntiviral activity,a nd nanotechnology. [2] So far,RNA-cleaving DNAzymes have been studied mostexten-sively,a nd they often require metal ions for activity.D NAzymes requiring ad iverse range of metal ions have been reported to date, such as Pb 2 + , [3] Zn 2 + , [4] UO 2 2 + , [5] Hg 2 + , [6] Cd 2 + , [7] trivalent lanthanides, [8] and other metals. [1c, 9]

Probing Metal-Dependent Phosphate Binding for the Catalysis of the 17E DNAzyme

“…Some of the most extensively studied DNAzymes contain the 8–17 motif. − Extensive biochemical assays have been carried out using various metal cofactors, ,,− DNAzyme core mutation, − and a change in the substrate cleavage junction . The same motif has been reported for many different selection conditions by multiple research groups.…”

Improving the Sensitivity of a Mn(II)-Specific DNAzyme for Cellular Imaging Sensor through Sequence Mutations

“…Based on these components, we analyzed the next-generation sequencing (NGS) sequences from the in vitro selected DNAzyme library. Since the secondary structure of the 11-5 DNAzyme is similar to that of the 8-17 DNAzyme, we have also compared known variants of the 8-17 DNAzyme. ,− From these analyses, we identified potential nucleotides in the catalytic domain of the 11-5 DNAzyme and performed site-specific mutations targeting these nucleotides.…”

Interfacing Catalytic DNA with Nanomaterials

“…[40] Recent studies showed that 17E might be a special form of GR5. [41,42] More recently, a Na + -specific DNAzyme named NaA43 was reported (Figure 1D), which works without the need of any divalent metal ion. [43] Its specificity for Na + was from an aptamer motif, [44][45][46][47] and a few other DNAzymes also contain this aptamer motif.…”