Site-specific cleavage of HCV genomic RNA and its cloned core and NS5B genes by DNAzyme.
OBJECTIVE
The 9600 nt hepatitis C virus (HCV) genomic RNA has only one internal ribosome entry site (IRES) for translation to a single polyprotein. In search of nucleic acid-based antiviral agents, two 10-23 DNAzymes were designed to cleave the RNA in IRES and RNA dependent RNA polymerase (RDRP/NS5B) regions to prevent translation and replication of HCV RNA.


METHODS
In vitro cleavage of HCV RNA by IRES specific DNAzyme, CDz and NS5B specific DNAzyme, NDz was carried out using HCV genomic RNA and in vitro synthesized runoff transcripts of core and NS5B genes. Cleavage of core and NS5B mRNAs by DNAzyme (Dz) in HepG2 cells was assessed by reverse transcription polymerase chain reaction (RT-PCR) using RNA from cells co-transfected with cloned core or NS5B gene and its respective DNAzyme. Suppression of core or NS5B protein expression due to mRNA cleavage by Dz in co-transfected cells was determined by Western blot analysis and fluorescence intensity of fluorescent-tagged expressed protein. Reduction of NS5B protein activity in NDz co-transfected cells was determined by enzymatic assays.


RESULTS
The designed CDz and NDz cleaved HCV genomic RNA and their respective in vitro generated transcripts. Both mRNA and protein expressions of core or NS5B from their cloned genes reduced substantially when co-transfected with respective Dz. Reduction of RDRP expression by NDz was accompanied with its reduced enzyme activity. Increased RNA cleavage, inhibition of protein expression, and reduction of RDRP activity were observed on increasing Dz concentration.


CONCLUSIONS
Core and NS5B targeted DNAzymes can be used in controlling the replication of HCV RNA.
Kumar
Deepak
D
Institute of Genomics and Integrative Biology, Delhi University Campus, Delhi, India.
Chaudhury
Indrajit
I
Kar
Premashis
P
Das
Rakha H
RH
D020121:5' Untranslated Regions; D000998:Antiviral Agents / Q000378:metabolism / Q000494:pharmacology*; D045744:Cell Line, Tumor; D021881:DNA, Catalytic / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D015967:Gene Expression Regulation, Viral / Q000187:drug effects; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development; D006801:Humans; D014176:Protein Biosynthesis / Q000187:drug effects; D012333:RNA, Messenger / Q000096:biosynthesis*; D012367:RNA, Viral / Q000096:biosynthesis*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014162:Transfection; D014758:Viral Core Proteins / Q000096:biosynthesis* / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis* / Q000235:genetics; D014779:Virus Replication / Q000187:drug effects; D012324:RNA-Dependent RNA Polymerase
D020121:5' Untranslated Regions; D000998:Antiviral Agents; D021881:DNA, Catalytic; D004277:DNA, Single-Stranded; D012333:RNA, Messenger; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins; C402582:RNA-cleaving DNA 10-23; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase