Objective: To explore the cleaving and inhibitory activity of hepatitis C virus ( HCV) -specific deoxyri-bozymes (DRz) at both molecular and transgeneic cellular levels. Methods: According to the secondary structure o...Objective: To explore the cleaving and inhibitory activity of hepatitis C virus ( HCV) -specific deoxyri-bozymes (DRz) at both molecular and transgeneic cellular levels. Methods: According to the secondary structure of HCV 5'-noncoding region (5'-NCR) and the sites characterized with 5'…Y ↓ R…3'(Y = A/G,R = U/C) , HCV-spe-cific naive deoxyribozymes were designed and named DRz-232, DRz-127, DRz-84, DRz1, and the phosphorothioate deoxyribozymes (PSDRz) and mutated phosphorothioate deoxyribozymes (MPSDRz) were also designed. HCV RNA 5'-NCR was transcribed in vitro from linearized plasmid pHCV-neo and radiolabelled at its 5'-end. DRz, PSDRz or MPSDRz was respectively mixed with the substrate RNA and incubated under appropriate conditions, the cleaved products were displayed by 8% denaturated polyacrylamide gel electrophoresis (PAGE) and autoradiography, and the optical density of each band was measured to calculate cleavage rates. After that, every kind of DRz was added respectively to the cultured transgeneic HepG2 cells containing luciferase gene controlled by HCV 5'-NCR. The cells were lysed at intended time points and the activity of luciferase was measured with chemiluminescence method for calculating inhibition rates. Results: After incubated for 90 min in vitro, the cleavage rates of DRz-127, PSDRz-127, DRz1 and PS-DRz1 reached 32.6% , 30. 8% , 24. 3% and 21. 5% , respectively. No cleavage product was observed in any MPSDRz. DRz-127, PSDRz-127, DRz1 and PSDRzl had an inhibitory rate of 53. 2% , 50. 6% , 44. 7% and 43. 3% respectively in transgeneic HepG2 cells in the first 24 h when the final dose of the DRz was 0. 5μmol/L, higher than that of the corresponding MPSDRz. There was no significant difference between the inhibitory effect of each DRz and its PSDRz in HepG2 cells, but the inhibitory rate of DRz decreased more rapidly than that of the latter with the elapse of time. The results from transfection groups were significantly better than those of non-transfection groups. Conclusion: Rationally-designed HCV-specific deoxyribozymes are able to cleave target RNA at molecular level in vitro, and efficiently inhibit the expression of luciferase gene controlled by HCV 5'-NCR in transgeneic cells. Appropriate PSDRz may be more stable, and thus more suitable than the naive DRz in the application to cells. Introduction of the deoxyribozymes with transfection is more efficient than with direct delivering ways.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
DNA核酶是通过指数富集配体的系统进化技术(Systematic Evolution of Ligands by Exponential Enrichment,SELEX)筛选出来的具有催化活性的DNA分子。本文重点综述了DNA核酶的分类、筛选及主要应用。根据其催化作用方式,可将DNA核酶分为...DNA核酶是通过指数富集配体的系统进化技术(Systematic Evolution of Ligands by Exponential Enrichment,SELEX)筛选出来的具有催化活性的DNA分子。本文重点综述了DNA核酶的分类、筛选及主要应用。根据其催化作用方式,可将DNA核酶分为切割类、连接类、磷酸化类和其他类,其中切割类和连接类占的比例较大。目前DNA核酶的底物主要还是以核酸居多。DNA核酶的筛选主要是采用生物素-链霉亲和素法和聚丙烯酰胺凝胶电泳法来分离有催化活性的DNA分子。目前DNA核酶在金属离子检测、基因治疗和DNA分子加密系统中都呈现出较好的应用前景。展开更多
文摘Objective: To explore the cleaving and inhibitory activity of hepatitis C virus ( HCV) -specific deoxyri-bozymes (DRz) at both molecular and transgeneic cellular levels. Methods: According to the secondary structure of HCV 5'-noncoding region (5'-NCR) and the sites characterized with 5'…Y ↓ R…3'(Y = A/G,R = U/C) , HCV-spe-cific naive deoxyribozymes were designed and named DRz-232, DRz-127, DRz-84, DRz1, and the phosphorothioate deoxyribozymes (PSDRz) and mutated phosphorothioate deoxyribozymes (MPSDRz) were also designed. HCV RNA 5'-NCR was transcribed in vitro from linearized plasmid pHCV-neo and radiolabelled at its 5'-end. DRz, PSDRz or MPSDRz was respectively mixed with the substrate RNA and incubated under appropriate conditions, the cleaved products were displayed by 8% denaturated polyacrylamide gel electrophoresis (PAGE) and autoradiography, and the optical density of each band was measured to calculate cleavage rates. After that, every kind of DRz was added respectively to the cultured transgeneic HepG2 cells containing luciferase gene controlled by HCV 5'-NCR. The cells were lysed at intended time points and the activity of luciferase was measured with chemiluminescence method for calculating inhibition rates. Results: After incubated for 90 min in vitro, the cleavage rates of DRz-127, PSDRz-127, DRz1 and PS-DRz1 reached 32.6% , 30. 8% , 24. 3% and 21. 5% , respectively. No cleavage product was observed in any MPSDRz. DRz-127, PSDRz-127, DRz1 and PSDRzl had an inhibitory rate of 53. 2% , 50. 6% , 44. 7% and 43. 3% respectively in transgeneic HepG2 cells in the first 24 h when the final dose of the DRz was 0. 5μmol/L, higher than that of the corresponding MPSDRz. There was no significant difference between the inhibitory effect of each DRz and its PSDRz in HepG2 cells, but the inhibitory rate of DRz decreased more rapidly than that of the latter with the elapse of time. The results from transfection groups were significantly better than those of non-transfection groups. Conclusion: Rationally-designed HCV-specific deoxyribozymes are able to cleave target RNA at molecular level in vitro, and efficiently inhibit the expression of luciferase gene controlled by HCV 5'-NCR in transgeneic cells. Appropriate PSDRz may be more stable, and thus more suitable than the naive DRz in the application to cells. Introduction of the deoxyribozymes with transfection is more efficient than with direct delivering ways.
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.
文摘DNA核酶是通过指数富集配体的系统进化技术(Systematic Evolution of Ligands by Exponential Enrichment,SELEX)筛选出来的具有催化活性的DNA分子。本文重点综述了DNA核酶的分类、筛选及主要应用。根据其催化作用方式,可将DNA核酶分为切割类、连接类、磷酸化类和其他类,其中切割类和连接类占的比例较大。目前DNA核酶的底物主要还是以核酸居多。DNA核酶的筛选主要是采用生物素-链霉亲和素法和聚丙烯酰胺凝胶电泳法来分离有催化活性的DNA分子。目前DNA核酶在金属离子检测、基因治疗和DNA分子加密系统中都呈现出较好的应用前景。
