Novel mono- and di-DNA-enzymes targeted to cleave TAT or TAT-REV RNA inhibit HIV-1 gene expression.
The regulatory proteins TAT and REV play a very important role in the transcription and replication of HIV-1. In order to seHIV-01lectively down regulate the expression of these genes we synthesized several mono- and one di-DNA-enzyme against the TAT or TAT-REV RNA. Several mono-DNA-enzymes possessing the 10-23 catalytic motif were assembled that were targeted to the predicted loop region of TAT or TAT/REV RNA. The cleavage efficiency of each mono-DNA-enzyme was variable and independent of the size of the predicted loop structure of the target RNA. DNA-enzyme targeted against the largest loop region cleaved the substrate RNA poorly. Mono-DNA-enzyme-5944 that targets only the TAT region cleaved the substrate poorly but the DNA-enzyme-5970 that overlaps TAT and REV showed potent cleavage activity. The two DNA-enzymes, when placed in tandem, cleaved the target RNA at multiple sites that were specific for the two mono-DNA-enzymes. Only Dz-5970 retained the ability to cleave the target RNA specifically at simulated physiological conditions. They were able to inhibit HIV-1 specific genes efficiently when introduced into a mammalian cell. The extent of inhibition correlated with their cleavage efficiency obtained at standard conditions of cleavage. Although DNA-enzyme-5970 showed the highest reduction (approximately 90%), other DNA-enzymes (mono-DNA-enzyme-5944 and the di-DNA-enzyme) also showed reduction to an extent of 60 and 80% respectively. The inhibitory effect of the DNA-enzyme could be overcome by providing HIV-1 TAT to the cells.
Unwalla
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Laboratory of Virology, National Institute of Immunology, JNU Campus, Aruna Asaf Ali Marg, New Delhi-110067, India.
Banerjea
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D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D002460:Cell Line; D002522:Chlorocebus aethiops; D021881:DNA, Catalytic; D004277:DNA, Single-Stranded / Q000378:metabolism*; D015870:Gene Expression; D015694:Gene Products, rev / Q000235:genetics*; D015696:Gene Products, tat / Q000235:genetics*; D016655:HIV Core Protein p24 / Q000235:genetics; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D015636:Magnesium Chloride; D008969:Molecular Sequence Data; D016296:Mutagenesis; D012367:RNA, Viral / Q000378:metabolism*; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus
D021881:DNA, Catalytic; D004277:DNA, Single-Stranded; D015694:Gene Products, rev; D015696:Gene Products, tat; D016655:HIV Core Protein p24; D012367:RNA, Viral; C402582:RNA-cleaving DNA 10-23; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus; D015636:Magnesium Chloride