Background: Several types of RNA enzymes (ribozymes) have been identified in biological systems and generated in the laboratory. Considering the variety of known RNA enzymes and the similarity of DNA and RNA, it is reasonable to imagine that DNA might be able to function as an enzyme as well. No such DNA enzyme has been found in nature, however. We set out to identifiy a metal-dependent DNA enzyme using in vitro selection methodology.

Results: Beginning with a population of 1Or4 DNAs containing 50 random nucleotides, we carried out five successive rounds of selective amplification, enriching for individuals that best promote the Pb2+-dependent cleavage of a target ribonucleoside 3’-OP bond embedded within an otherwise all-DNA sequence. By the fifth round, the population as a whole carried out this reaction at a rate of 0.2 nun-*. Based on the sequence of 20 individuals isolated from this population, we designed a simplified version of the catalytic domain that operates in an intermolecular context with a turnover rate of 1 miri ‘. This rate is about 105-fold increased compared to the uncatalyzed reaction.

Conclusions: Using in vi&0 selection techniques, we obtained a DNA enzyme that catalyzes the Pv+-dependent cleavage of an RNA phosphoester in a reaction that proceeds with rapid turnover. The catalytic rate compares favorably to that of known RNA enzymeswe expect that other examples of DNA enzymes will soon be forthcoming.