Selective inactivation of a target gene by antisense mechanisms is an important biological tool to delineate specific functions of the gene product. Approaches mediated by ribozymes and RNA-cleaving DNA enzymes (DNA enzymes) are more attractive because of their ability to catalytically cleave the target RNA. DNA enzymes have recently gained a lot of importance because they are short DNA molecules with simple structures that are expected to be stable to the nucleases present inside a mammalian cell. We have designed a strategy to identify accessible cleavage sites in HIV-1 gag RNA from a pool of random DNA enzymes, and for isolation of DNA enzymes. A pool of random sequences (all 29 nucleotides long) that contained the earlier-identified 10Ő23 catalytic motif were tested for their ability to cleave the target RNA. When the pool of random DNA enzymes was targeted to cleave between any A and U nucleotides, DNA enzyme 1836 was identified. Although several DNA enzymes were identified using a pool of DNA enzymes that was completely randomized with respect to its substrate-binding properties, DNA enzyme-1810 was selected for further characterization. Both DNA enzymes showed target-specific cleavage activities in the presence of Mg2+ only. When introduced into a mammalian cell, they showed interference with HIV-1-specific gene expression. This strategy could be applied for the selection of desired target sites in any target RNA.

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