Potent knock down of HIV-1 replication by targeting HIV-1 Tat/Rev RNA sequences synergistically with catalytic RNA and DNA.
OBJECTIVE
Ribozymes (Rzs) and DNA-enzymes (Dzs) possess the ability to prevent gene expression by cleaving target RNA in a catalytic and sequence-specific manner. Although Rzs or Dzs have been used earlier for HIV-1 gene suppression, the present study explored the possibility of using catalytic RNA and DNA simultaneously in a synergistic manner with the hope that this novel approach will allow more potent inhibition for a longer duration.


METHODS
In order to achieve long-term inhibition of HIV-1 replication, a novel non-GUX hammerhead Rz was designed by standard recombinant DNA technology and cloned it under the powerful CMV promoter containing expression vector. A 10-23 catalytic motif containing Dz that was targeted against the conserved second exon of HIV-1 Tat/Rev region was also assembled.


RESULTS
Both Rz and Dz possessed sequence-specific cleavage activities individually and simultaneously cleaved target RNA in a synergistic manner under the same in vitro cleavage conditions. These catalytic molecules inhibited HIV-1 replication in macrophages individually and exhibited potent inhibitory effects when used in combination.


CONCLUSIONS
The combination strategy described here can be widely used against any target RNA to achieve more effective gene inhibition that exploits the simultaneous sequence-specific cleavage potentials of catalytic RNA and DNA.
Sood
Vikas
V
Laboratory of Virology, National Institute of Immunology, JNU Campus, Aruna Asaf Ali Marg, New Delhi, India.
Unwalla
Hoshang
H
Gupta
Nidhi
N
Chakraborti
Samitabh
S
Banerjea
Akhil C
AC
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D021881:DNA, Catalytic; D005786:Gene Expression Regulation; D018390:Gene Targeting; D015757:Genes, rev; D015756:Genes, tat; D005818:Genetic Engineering; D015316:Genetic Therapy / Q000379:methods*; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008264:Macrophages / Q000821:virology; D008969:Molecular Sequence Data; D016337:RNA, Catalytic; D016364:Reading Frames; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014779:Virus Replication / Q000235:genetics
D021881:DNA, Catalytic; D016337:RNA, Catalytic