In vitro-selected RNA cleaving DNA enzymes from a combinatorial library are potent inhibitors of HIV-1 gene expression.
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 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 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 Mg(2+) 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.
Sriram
B
B
Laboratory of Virology, National Institute of Immunology, JNU Campus, Aruna Asaf Ali Marg, New Delhi-110067, India.
Banerjea
A C
AC
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019556:COS Cells; D002384:Catalysis / Q000187:drug effects; D003001:Cloning, Molecular; D004261:DNA Replication; D021881:DNA, Catalytic / Q000737:chemistry / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral; D015723:Gene Library; D017930:Genes, Reporter / Q000235:genetics; D015750:Genes, gag / Q000235:genetics; D015497:HIV-1 / Q000235:genetics* / Q000502:physiology; D015636:Magnesium Chloride / Q000494:pharmacology; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014779:Virus Replication
D021881:DNA, Catalytic; D012367:RNA, Viral; D015636:Magnesium Chloride