An in vitro selection procedure was used to develop a DNA enzyme that can be made to cleave almost any targeted RNA substrate under simulated physiological conditions. The enzyme is comprised of a catalytic domain of 15 deoxynucleotides, f lanked by two substrate-recognition domains of seven to eight deoxynucleotides each. The RNA substrate is bound through Watson-Crick base pairing and is cleaved at a particular phosphodiester located between an unpaired purine and a paired pyrimidine residue. Despite its small size, the DNA enzyme has a catalytic efficiency (k cat ͞K m ) of Ϸ10 9 M ؊1 ⅐min ؊1 under multiple turnover conditions, exceeding that of any other known nucleic acid enzyme. Its activity is dependent on the presence of Mg 2؉ ion. By changing the sequence of the substrate-recognition domains, the DNA enzyme can be made to target different RNA substrates. In this study, for example, it was directed to cleave synthetic RNAs corresponding to the start codon region of HIV-1 gag͞pol, env, vpr, tat, and nef mRNAs.DNA has long been regarded as a passive molecule, ideally suited for carrying genetic information but structurally monotonous and therefore functionally impoverished. After the discovery of catalytic RNA (1, 2), it became clear that nucleic acid molecules of a particular sequence and 3-dimensional structure are able to carry out specific chemical reactions, often with an efficiency comparable to that of protein enzymes (3). In recent years, the first examples of DNA enzymes have appeared, each having been obtained by in vitro selection methodology (4-7). Although it is remarkable that DNA can have catalytic activity, all of the DNA enzymes generated to date have little utility in a biological context. This is in contrast to some of the naturally occurring RNA enzymes, such as the ''hammerhead'' and ''hairpin'' ribozymes, which have been used to cleave and thereby inactivate target viral and messenger RNAs (reviewed in ref. 8).We sought to develop a DNA enzyme that could be made to cleave almost any RNA substrate, efficiently and specifically under physiological conditions. Such a molecule could be used to inactivate a target RNA, probe a structured RNA, or assist in the manipulation of recombinant RNA. Compared with synthetic RNA enzymes, DNA enzymes are easier to prepare and less sensitive to chemical and enzymatic degradation. In a previous study, DNA was shown to catalyze the Mg 2ϩ -dependent cleavage of an RNA phosphoester embedded within an otherwise all-DNA substrate (6). Although that DNA enzyme was unable to cleave an all-RNA substrate, its properties suggested that a DNA enzyme with general purpose RNA cleavage activity might be attainable.Using in vitro selection, we carried out an extensive search of DNA sequences, seeking molecules that best met the following criteria: (i) ability to cleave RNA with multiple turnover under simulated physiological conditions (e.g., 2 mM MgCl 2 ͞150 mM KCl, pH 7.5, 37ЊC); (ii) ability to recognize the RNA substrate through Watson-Crick base pairing; ...

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Molecular self-assembly offers a means of spontaneously forming complex and well-defined structures from simple components. The specific bonding between DNA base pairs has been used in this way to create DNA-based nanostructures and to direct the assembly of material on the subnanometre to micrometre scale. In principle, large-scale clonal production of suitable DNA sequences and the directed evolution of sequence lineages towards optimized behaviour can be realized through exponential DNA amplification by polymerases. But known examples of three-dimensional geometric DNA objects are not amenable to cloning because they contain topologies that prevent copying by polymerases. Here we report the design and synthesis of a 1,669-nucleotide, single-stranded DNA molecule that is readily amplified by polymerases and that, in the presence of five 40-mer synthetic oligodeoxynucleotides, folds into an octahedron structure by a simple denaturation-renaturation procedure. We use cryo-electron microscopy to show that the DNA strands fold successfully, with 12 struts or edges joined at six four-way junctions to form hollow octahedra approximately 22 nanometres in diameter. Because the base-pair sequence of individual struts is not repeated in a given octahedron, each strut is uniquely addressable by the appropriate sequence-specific DNA binder.

All life that is known to exist on Earth today and all life for which there is evidence in the geological record seems to be of the same form--one based on DNA genomes and protein enzymes. Yet there are strong reasons to conclude that DNA- and protein-based life was preceded by a simpler life form based primarily on RNA. This earlier era is referred to as the 'RNA world', during which the genetic information resided in the sequence of RNA molecules and the phenotype derived from the catalytic properties of RNA.

The discovery of RNA enzymes has, for the first time, provided a single molecule that has both genetic and catalytic properties. We have devised techniques for the mutation, selection and amplification of catalytic RNA, all of which can be performed rapidly in vitro. Here we describe how these techniques can be integrated and performed repeatedly within a single reaction vessel. This allows evolution experiments to be carried out in response to artificially imposed selection constraints. We worked with the Tetrahymena ribozyme, a self-splicing group I intron derived from the large ribosomal RNA precursor of Tetrahymena thermophila that catalyses sequence-specific phosphoester transfer reactions involving RNA substrates. It consists of 413 nucleotides, and assumes a well-defined secondary and tertiary structure responsible for its catalytic activity. We selected for variant forms of the enzyme that could best react with a DNA substrate. This led to the recovery of a mutant form of the enzyme that cleaves DNA more efficiently than the wild-type enzyme. The selected molecule represents the discovery of the first RNA enzyme known to cleave single-stranded DNA specifically.