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

Abstract

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.