Establishment Of A Real-time Monitoring System For Kinetic Characterization Of RNA-cleaving DNAzyme/LNAzyme And Inhibition Of Hepatitis C Virus Gene Expression By DNAzyme/LNAzyme In Vitro

DNA in biological systems has long been considered a passive molecule carrying genetic information. Single-stranded DNA can also fold into well-defined, sequence-dependent tertiary structures, specifically bind to a variety of target molecules, and exhibit catalytic activities similar to those of ribozymes or protein enzymes. DNAzymes are synthetic, singlestranded DNA catalysts that can be engineered to bind to their complementary sequence in a target messenger RNA (mRNA) through Watson-Crick base pairing have been generated using in vitro selection methodology. A general model for the DNAzymes has been proposed as 10-23 DNAzyme, which has a catalytic domain of 15 deoxyribonucleotides, flanked by two substrate-recognition domains of seven to eight deoxyribonucleotides each. It was derived from the 23rd clone obtained after the 10th round of selective amplification. According to the specific binding between recognition domains and RNA substrate, the selected target RNA is cleaved at specific phosphodiester bond, which is located between an unpaired purine and a paired pyrimidine residue. By appropriately designing the sequences of the two substrate-recognition domains, the molecule can be made to cleave virtually any RNA that contains a purine-pyrimidine junction. Incorporation of an optimal number of LNA(locked nucleic acid) monomers into the binding arms, referred as LNAzyme, could significantly enhance helical thermostability and improve cleavage kinetics.DNAzymes/LNAzyme can be designed specifically to recognize the AU nts of the start codon, which make it promising candidates for potential applications in functional genomics and gene therapy. Using this kind of DNAzymes/LNAzyme to inhibit the gene expressions of some RNA viruses have ever been reported including our previous study. In addition, their capacity to block of a diverse range of pathologies in animal models also suggests a potential use of DNAzymes/LNAzyme as therapeutic tools.Hepatitis C virus (HCV) is positive single-stranded RNA virus within the Flaviviridae family. The genome of HCV comprises approximate 9,400 nucleotides(nt) containing 5' untranslating region (UTR)% structural region(C,E)> non structural region(NS) and 3' untranslating region (UTR). HCV RNA contains an open reading frame(ORF) that encodes a large polypeptide precursor beginning with the first in-frame methionine codon (AUG) within region C. Various viral proteins are released by the action of a host-cell signal peptidase and NS3 proteinase. Therefore, specific gene-blocking sites selected within HCV ORF could be cleaved by DNAzymes/LNAzyme, which would effectively block the expression of HCV.This study demonstrated the inhibition effects of HCV gene expression mediated by DNAzymes/LNAzyme specific to HCV ORF AUG in vitro from two aspects. Firstly, a real-time monitoring system for kinetic characterization of DNAzyme/LNAzyme was successfully established in the first time. Using this system, specific DNAzyme and LNAzyme were screened. Then, using enhanced green fluorescence protein (EGFP) as reporting molecule, the inhibition effects of HCV gene expression were observerd in HepG2 cells and HCV-UTR/C126-EGFP stably-expressing HepG2 cells, respectively.Taken together, the results in this report fully show that DNAzymes/LNAzyme specific to HCV ORF AUG could inhibit HCV gene expression. The real-time monitoring system for kinetic characterization of RNA-cleaving DNAzyme/LNAzyme established in the first time here were undoubtedly sure to allow kinetic analysis in a rapid, accurate and high-throughput way.Part I Establishment of a real-time monitoring system for kinetic characterization of RNA-cleaving DNAzyme/LNAzymeObjective To Establishment of a real-time monitoring system for kinetic characterization of RNA-cleaving DNAzyme/LNAzyme.Methods1. DNAzyme/LNAzyme named Dz-HCV-9 and Lz-HCV-9 specific to HCVORF AUG were designed and synthesized. Dz-HCV-Mis-9 with mismatched substrate-recognition domains, Dz-HCV-Mut-9 with mutant catalytic domains, antisense oligonucleotide ASON and nonsense oligonucleotide NSON were synthesized respectively as controls.2. A chimeric oligonucleotide of 25nt containing both RNA and DNA bases was designed and synthesized. The reporter FAM/BHQ was incorporated at the 5' and 3' end, respectively.3. Under simulated physiological conditions (37"C), kinetic characterization of RNA-cleaving DNAzyme/LNAzyme was analyzed in a real-time way.4. Factors that influencing DNAzyme/LNAzyme cleavage were analyzed Results1. Dz-HCV-9 and Lz-HCV-9 specific to HCV ORF AUG could cleave target RNA at A?U site, a continuous change of fluorescence intensity was monitored. While the control oligonucleotides couldn't cleave RNA, there were no change of fluorescence intensity.2. Factors that influencing DNAzyme/LNAzyme cleavage concluded different substrate-recognition domain, Mg2+concentration and pH.ConclusionA real-time monitoring system for kinetic characterization of RNA-cleaving DNAzyme/LNAzyme was successfully established in the first time.Part II Inhibition of hepatitis C virus gene expression by DNAzyme/LNAzyme in HepG2 cellsObjective To study the DNAzyme/LNAzyme-mediated inhibition of HCV gene expression in HepG2 cells.Methods1. A HCV-UTR/C126-EGFP fusion protein eukaryotic expression plasmidwas constructed using recombinational DNA techniques and identified by different methods including PCR> digestion and sequencing.2. HC V-UTR/C 126-EGFP stably-expressing HepG2 cells were selected by the ability of resistance to G418 and isolated with a limited dilution and identified by different methods including PCR> RT-PCR^ Southern blot, and FCM.3. Inhibition effects were observed by fluorescent microscopy and detected by FCM and semi-quantitative RT-PCR in transient transfected HepG2 cells, and also detected by FCM in HCV-UTR/C126-EGFP stably-expressing HepG2 cells.Results1. The HCV-UTR/C126-EGFP fusion protein eukaryotic expression plasmid was successfully constructed.2. The HCV-UTR/C126-EGFP stably-expressing HepG2 cells were successfully obtained.3. Inhibition effects were successfully observed and detected in transient transfected HepG2 cells and HCV-UTR/C126-EGFP stably-expressing HepG2 cells.ConclusionDNAzymes/LNAzyme specific to HCV ORF AUG could inhibit HCV gene expression. They could be new tools for HCV gene therapy.