A comparison of two classic Pb2+-dependent RNA-cleaving DNAzymes

Pb2+ is a very important metal cofactor in DNAzyme catalysis. GR5 is the first reported DNAzyme, and 17E is the most thoroughly studied. Both have the highest activity with Pb2+ and are by far the fastest RNA-cleaving DNAzymes. GR5 reacts only with Pb2+ while 17E is also active with a number of other divalent metal ions. It is also interesting to note that Pb2+ shows activity with most RNA-cleaving DNAzymes. To understand these Pb2+-dependent DNAzymes and the occurrence of DNAzyme sequences, herein systematic mutation studies are performed on GR5. A comparison with 17E is also made. The A6, G7, C13, and G14 positions in 17E have been previously established to be crucial and we report A6, G7, C14, and G15 in GR5 to have the same role. The guanine at the cleavage site dinucleotide junction of the substrate strand is also mutated to hypoxanthine, 2-aminopurine, and adenine. Again, both enzymes show the same trend of activity change. Our results suggest that both DNAzymes have a similar binding pocket for Pb2+. The reason for Pb2+ being active in many DNAzymes is attributed to its simple binding motif requirement. Finally, we propose that 17E is a special form of GR5. They both have the simple sequence requirements needed for Pb2+-dependent activity, but 17E has additional motifs making it active also with other divalent metal ions.