A DNA structure is described that can cleave single-stranded DNA oligonucleotides in the presence of ionic copper. This ''deoxyribozyme'' can self-cleave or can operate as a bimolecular complex that simultaneously makes use of duplex and triplex interactions to bind and cleave separate DNA substrates. Bimolecular deoxyribozyme-mediated strand scission proceeds with a k obs of 0.2 min ؊1 , whereas the corresponding uncatalyzed reaction could not be detected. The duplex and triplex recognition domains can be altered, making possible the targeted cleavage of single-stranded DNAs with different nucleotide sequences. Several small synthetic DNAs were made to function as simple ''restriction enzymes'' for the site-specific cleavage of single-stranded DNA.DNA in biological systems exists primarily in duplex form where it serves almost exclusively as a storage system for genetic information. Outside the confines of cells, DNA in its single-stranded form can be made to perform both molecular recognition and catalysis-biochemical operations that were until recently thought to be possible only with macromolecules made of protein or RNA. For example, a number of DNA ''aptamers'' (1) can be made that function as ligands for proteins or as highly specific receptors for small organic molecules (2-4). In addition, certain single-stranded DNAs act as artificial enzymes (4-6), catalyzing such chemical reactions as phosphoester transfer (7-12), phosphoester formation (13), porphyrin metalation (14), phosphoramidate cleavage (15), and DNA cleavage (16). Most likely, DNA can be made to perform a much broader repertoire of catalytic activities (6).These capabilities of DNA conceivably can be exploited to create a variety of structured DNAs that perform useful tasks, either in vitro or in vivo, involving molecular recognition and catalysis. Such functional DNAs offer several advantages, including ease of synthesis and chemical stability, that might make attractive properties for polymers that serve as artificial receptors or as biocatalysts for various applications. Characteristics such as thermostability and solvent͞solute preferences could be conferred upon deoxyribozymes by using both rational and combinatorial methods of molecular design. Catalytic DNAs may offer distinct advantages over natural protein enzymes for operation under nonbiological conditions.In a previous study (16), we reported the isolation of two distinct types of deoxyribozymes (classes I and II) that undergo oxidative self-cleavage in the presence of copper ions. By using in vitro selection (17), class II self-cleaving DNAs have been further optimized for catalytic function, and the most active structure obtained from this process has been engineered to act as a ''restriction endonuclease'' for single-stranded DNA substrates. MATERIALS AND METHODSOligonucleotides. Synthetic DNAs were prepared by automated chemical synthesis (Keck Biotechnology Resource Laboratory, Yale University) and were purified by denaturing (8 M urea) PAGE prior to use. Double-stran...