BACKGROUND: 10-23 DNA enzyme is one kind of de-oxyribozymes for RNA cleavage. The inhibition effects of 10-23 DNA enzyme on the expression of the HBV C gene in HepG2. 2. 15 cells were demonstrated previously. The aim ...BACKGROUND: 10-23 DNA enzyme is one kind of de-oxyribozymes for RNA cleavage. The inhibition effects of 10-23 DNA enzyme on the expression of the HBV C gene in HepG2. 2. 15 cells were demonstrated previously. The aim of this study was to further explore the cleavage activities of 10-23 DNA enzyme targeting at HBV C gene mRNA in vitro. METHODS: 10-23 DNA enzyme named Drz-HBV-C-9 specific to HBV C gene ORF A1816UG was designed and synthesized. HBV C gene mRNA was obtained by the in vitro transcription method. Cleavage activities of Drz-HBV-C-9 were observed in vitro. Values of kinetic parameters including Km,Kcat and Kcat/Km were calculated accordingly. RESULTS: Under the certain cleavage conditions, Drz-HBV-C-9 could efficiently cleave target mRNA at specific sites in vitro. Cleavage products of 109nt plus 191nt were obtained. The kinetic parameters, Km,Kcat and Kcat/ Km for Drz-HBV-C-9, were 1.4 ×10-9 mol, 1.6 min-1 and 1.1 × 109 mol-1 · min-1, respectively. CONCLUSIONS: 10-23 DNA enzyme targeting at HBV C gene mRNA possesses specific cleavage activities in vitro. This would be a potent antiviral strategy with respect to HBV gene therapy.展开更多
Objective To prepare fluorescent silica nanoparticles(FSNPs) carrying 10-23 deoxyribozymes(10-23DRzs) and to evaluate their inhibitory effect on human hepatitis B virus(HBV).Methods The FSNPs were prepared by microemu...Objective To prepare fluorescent silica nanoparticles(FSNPs) carrying 10-23 deoxyribozymes(10-23DRzs) and to evaluate their inhibitory effect on human hepatitis B virus(HBV).Methods The FSNPs were prepared by microemulsion method and further modified by NaCl.Their diameter and the Zeta potential were tested.The HBV-specific 10-23DRzs were designed according to the sequences of S and C genes of HBV.The 10-23DRzs were connected to FSNPs,which constituted the FSNPs-DNA.The connecting efficiency and the protective effect of nanoparticles on 10-23DRzs were tested by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).The HepG2.2.15 cells were transfected by FSNPs-DNA,of which the inhibitory effects on HBsAg and HBeAg were analyzed by enzyme-linked immunosorbent assay(ELISA).Results The nanoparticles were spherical and uniform in size,with a diameter of 220 nm and the surface Zeta potential of +15.2 mV.The combination of DNA and FSNPs effectively protected DNA from nuclease degradation.Transfection of FSNPs-DNA significantly inhibited the expression of HBV S and C genes compared to the liposome control group.Conclusion The FSNPs have been successfully prepared and efficiently connected to HBV-specific 10-23DRzs,which significantly inhibit the expression of HBV S and C genes in cell culture.展开更多
基金This study was supported by a grant from the National Natural Science Foundation of China (No. 30271183).
文摘BACKGROUND: 10-23 DNA enzyme is one kind of de-oxyribozymes for RNA cleavage. The inhibition effects of 10-23 DNA enzyme on the expression of the HBV C gene in HepG2. 2. 15 cells were demonstrated previously. The aim of this study was to further explore the cleavage activities of 10-23 DNA enzyme targeting at HBV C gene mRNA in vitro. METHODS: 10-23 DNA enzyme named Drz-HBV-C-9 specific to HBV C gene ORF A1816UG was designed and synthesized. HBV C gene mRNA was obtained by the in vitro transcription method. Cleavage activities of Drz-HBV-C-9 were observed in vitro. Values of kinetic parameters including Km,Kcat and Kcat/Km were calculated accordingly. RESULTS: Under the certain cleavage conditions, Drz-HBV-C-9 could efficiently cleave target mRNA at specific sites in vitro. Cleavage products of 109nt plus 191nt were obtained. The kinetic parameters, Km,Kcat and Kcat/ Km for Drz-HBV-C-9, were 1.4 ×10-9 mol, 1.6 min-1 and 1.1 × 109 mol-1 · min-1, respectively. CONCLUSIONS: 10-23 DNA enzyme targeting at HBV C gene mRNA possesses specific cleavage activities in vitro. This would be a potent antiviral strategy with respect to HBV gene therapy.
基金Supported by National Hi-tech Project of China(No.2007AA02-1803 and 2007AA021901)
文摘Objective To prepare fluorescent silica nanoparticles(FSNPs) carrying 10-23 deoxyribozymes(10-23DRzs) and to evaluate their inhibitory effect on human hepatitis B virus(HBV).Methods The FSNPs were prepared by microemulsion method and further modified by NaCl.Their diameter and the Zeta potential were tested.The HBV-specific 10-23DRzs were designed according to the sequences of S and C genes of HBV.The 10-23DRzs were connected to FSNPs,which constituted the FSNPs-DNA.The connecting efficiency and the protective effect of nanoparticles on 10-23DRzs were tested by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).The HepG2.2.15 cells were transfected by FSNPs-DNA,of which the inhibitory effects on HBsAg and HBeAg were analyzed by enzyme-linked immunosorbent assay(ELISA).Results The nanoparticles were spherical and uniform in size,with a diameter of 220 nm and the surface Zeta potential of +15.2 mV.The combination of DNA and FSNPs effectively protected DNA from nuclease degradation.Transfection of FSNPs-DNA significantly inhibited the expression of HBV S and C genes compared to the liposome control group.Conclusion The FSNPs have been successfully prepared and efficiently connected to HBV-specific 10-23DRzs,which significantly inhibit the expression of HBV S and C genes in cell culture.