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Here, we describe the characterization of new RNA‐cleaving DNAzymes that showed the highest catalytic efficiency at pH 4.0 to 4.5, and were completely inactive at pH values higher than 5.0. Importantly, these DNAzymes did not require any divalent metal ion cofactors for catalysis. This clearly suggests that protonated nucleic bases are involved in the folding of the DNAzymes into catalytically active structures and/or in the cleavage mechanism. The  trans ‐acting DNAzyme variants were also catalytically active. Mutational analysis revealed a conservative character of the DNAzyme catalytic core that underpins the high structural requirements of the cleavage mechanism. A significant advantage of the described DNAzymes is that they are inactive at pH values close to physiological pH and under a wide range of conditions in the presence of monovalent and divalent metal ions. These pH‐dependent DNAzymes could be used as molecular cassettes in biotechnology or nanotechnology, in molecular processes that consist of several steps. The results expand the repertoire of DNAzymes that are active under nonphysiological conditions and shed new light on the possible mechanisms of catalysis.

Characterization of Highly Efficient RNA‐Cleaving DNAzymes that Function at Acidic pH with No Divalent Metal‐Ion Cofactors