DNA‐based enzymes, also known as deoxyribozymes or DNAzymes, are single‐stranded DNA molecules with catalytic activity. DNAzymes do not exist in nature but can be isolated from random‐sequence DNA pools using in vitro selection. To date, many DNAzymes that collectively catalyze a diverse range of chemical transformations have been reported. Here, examples of new DNAzymes engineered to mimic some intriguing functions of naturally occurring protein‐based enzymes are discussed. This is followed by discussions of recent examples of a particular class of DNAzymes, known as “RNA‐cleaving DNAzymes”, that have been derived specifically so that their activity is strictly dependent on a given chemical or biological stimulus. Some unique ways to employ ligand‐responsive DNAzymes for the design of bioanalytical assays and biosensors are then highlighted. Being DNA molecules, DNAzymes have proven to be entirely compatible with DNA amplification. Several approaches are then discussed, which relay the activity of an analyte‐activated DNAzyme into the production of massive amounts of DNA amplicons, via “rolling circle amplification”, in biosensing applications designed to deliver very high levels of detection sensitivity.

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Molecular recognition elements with high specificity are of great importance for the study of molecular interactions, accurate diagnostics, drug design, and personalized medicine. Herein, a highly specific DNA aptamer for RNase H2 from Clostridium dif ficile (C. diff icile) was generated by SELEX and minimized to 40 nucleotides. The aptamer exhibits a dissociation constant (K d ) of 1.8 ± 0.5 nM and an inhibition constant (IC 50 ) of 7.1 ± 0.6 nM for C. diff icile RNase H2, both of which are 2 orders of magnitude better for the same enzyme from other control bacteria. The fluorescent version of the aptamer can distinguish C. diff icile from several other control bacteria in a cell lysate assay. This work demonstrates that a ubiquitous protein like RNase H2 can still be used as the target for the development of highly specific aptamers and the combination of the protein and the aptamer can achieve the recognition specificity needed for a diagnostic test and drug development.

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