In Vitro Selected RNA-Cleaving DNA Enzymes From Combinatorial Libraries

Abstract

The selective inactivation of a target gene by antisense mechanisms is an important biological tool in the delineation of gene functions. Ribozymes and RNA-cleaving DNA enzymes-mediated approaches are more attractive because of their ability to catalytically cleave the target RNA. DNA enzymes have recently gained great 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 human immunodeficiency virus-1 (HIV-1) gag RNA from a pool of random DNA enzymes, and for isolation of DNA enzymes. A pool of random sequences 29 nucleotides long that contained the previously identified 10–23 catalytic motif were tested for their ability to cleave the target RNA. When the pool of random DNA enzymes was targeted to cleave between A and U nucleotides, a DNA enzyme 1836 was identified. Although several DNA enzymes were identified using a pool of DNA enzyme that was completely randomized with respect to its substrate-binding properties, DNA enzyme-1810 was selected for further characterization. Both the DNA enzymes showed target-specific cleavage activities in the presence of Mg²⁺ only. These strategies could be applied for the selection of desired target sites in any target RNA.