Aptamer-based and DNAzyme-linked colorimetric detection of cancer cells

This paper reports a novel method to detect human leukemic lymphoblasts (CCRF-CEM cells). While the aptamer of the cancer cells was employed as the recognition element to target cancer cells, peroxidase-active DNAzyme was used as the sensing element to produce catalysis-induced colorimetric signals. The elegant architecture integrating the aptamer and DNAzyme made it feasible to detect cancer cells easily and rapidly by the color change of the substrate for DNAzyme. Experimental results showed that 500 cells can well indicate the cancer, while as control, 250,000 Islet Island Beta cells only show tiny signals, suggesting that the method proposed in this paper has considerable sensitivity and selectivity. Furthermore, since it does not require expensive apparatus, or modification or label of DNA chains, the method we present here is also cost-effective and conveniently operated, implying potential applications in future cancer diagnosis.

The effect of short hairpin RNA and DNAzyme gene therapy on latent membrane protein-1 expression in nasopharyngeal carcinoma / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To study the inhibitory effects of short hairpin RNA (pshLMP1) combined with DNAzyme or alone on latent membrane protein-1 (LMP1)expression in HNE1 cell lines.@*METHOD@#The pshLMP1 and DNAzymes were co-transfected with pEGFP-N1-1158 (a expression vector encoding LMP1 and EGFP fusion protein) into HNE1 cells, divided into 4 groups as positive control, RNA interference, combining and DNAzyme groups. The green fluorescence were analyzed and then the mRNA and protein of LMP1 gene were detected.@*RESULT@#The HNE1 cells expressing green fluorescence in combining group were significantly less than that in RNA interference group (P < 0.01); The inhibition efficiency of green fluorescence in combining group were 86. 31% Wp 88.88% ,which were higher than that in RNA interference group with 75.15%. The detection of LMP1 mRNA and proteins showed that combining group had a higher inhibition ability.@*CONCLUSION@#Combining gene therapy with pshLMP1 and DNAzyme is superior to pshLMP1 alone to inhibit LMP1 gene expression.

Influence of DNAzymes against cyclin D1 in tumor cell cycle / 生物医学工程学杂志

In this study, DNAzymes against cyclin D1 (cyclin D1-DRz) were designed according to the secondary structure of cyclin D1 mRNA which was computed with RNAdraw and Mfold. Cyclin D1-DRz were transfected into tumor cell line u251 and HeLa by oligofectamine. The expression of cyclin D1 was detected by RT-PCR. It was shown that the expression of cyclin D1 gene was suppressed obviously, and the expressions of other cell-cycle related genes such as cyclin E1, cyclin A1 and cyclin B1 were also declined. The cell cycle analysis of tumor cells tansfected with cyclin D1-DRz revealed an arrestment in the G0/G1 phase. In conclusion, the approach is effective and feasible for designing DNAzyme. Cyclin D1-DRz is useful for interfering with the cell cycle procession of tumor cells.

Research of 10-23 DNAZyme inhibit the expression of eIF4E genes / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To explore the possibility of 10-23DNAzyme becoming a new gene therapy for laryngeal carcinoma treatment at the cell level.@*METHOD@#Thiosthorothioate 10-23DNAzyme specific to eIF4E gene mRNA 1059 was designed and synthesized, and its inhibition effects on the expression of eIF4E gene in Hep-2 cells were observed.@*RESULT@#The expression of eIF4E gene was remarkable depressed after Hep-2 cells was transfected by DNAzyme. The level of inhibiting eIF4E in hep-2 cells transfected by DNAzyme was lower than that by only lipofectamine 2000 transfected and Hep-2.@*CONCLUSION@#The expression of eIF4E gene in Hep-2 cells 10-23DNAzyme can be highly blocked. It is a specific and effective gene therapeutic means.

Cleavage of in vitro transcripts of hepatitis B virus C gene by 10-23 DNA enzyme / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the cleavage activities of 10-23 DNA enzymes targeting at HBV C gene mRNA in vitro.</p><p><b>METHODS</b>10-23 DNA enzymes named DrzBC-7, DrzBC-8 and DrzBC-9 specific to HBV C gene ORF A1816UG were designed and synthesized. HBV C gene mRNA was obtained by in vitro transcription method. Cleavage activities were observed in vitro. The influence of MgCl2 concentration on RNA cleaving activity was examined with DrzBC-9. Values of kinetic parameters including Km, Kcat and Kcat/Km were calculated accordingly.</p><p><b>RESULT</b>Targeted substrate mRNA with the size of 300 nt was obtained by transcription in vitro. Under the certain cleavage conditions, DrzBC-7, DrzBC-8 and DrzBC-9 all efficiently cleaved target mRNA at specific sites in vitro. Cleavage products of 109 nt and 191 nt were obtained. No cleavage occurred without MgCl2. The most efficient cleavage was obtained at 150 mmol x L(-1) MgCl2. The efficiency of cleavage did not increase when the MgCl2 concentration was more than 200 mmol x L(-1). The kinetic parameters, Km, Kcat and Kcat/Km for DrzBC-9 were 1.4x10(-9) mol x L(-1), 1.6 min(-1) and 1.1x10(9) mol x L(-1) x min(-1), respectively.</p><p><b>CONCLUSION</b>10-23 DNA enzymes targeting at HBV C gene mRNA possess the specific cleavage activities in vitro.</p>

A study of 10-23 DNAzyme inhibiting the expression of hepatitis B virus genes / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To explore, on the cell level, the possibility of using 10-23DNAzyme, as a new genetherapy in treating hepatitis B.</p><p><b>METHODS</b>Phosthorothioate 10-23DRz (DRz-S) and 10-23DRz specific to HBV pre-C/C gene ORFA2031 were designed and synthesized, and the inhibition effects of 10-23DRz-S and 10-23DRz on the expression of HBV gene in HepG2 2.2.15 cells were observed.</p><p><b>RESULTS</b>The expression of HBV gene was remarkably depressed after 2.2.15 cells were transfected by DRz-S and DRz. The maximum inhibition was 93.75% and 90.26%. The inhibition effect was maintained for 96 hours, and the inhibition time of DRz-S was longer than that of DRz. The maximum inhibition of DRz-S was lower than that of DRz. The efficiency of inhibiting HBsAg and HBeAg in 2.2.15 cells transfected by DRz-S and DRz was higher than that by antisense oligonucleotides for the same target genes. It had no remarkable effect on the replication of HBV DNA and no toxicity to the 2.2.15 cells.</p><p><b>CONCLUSION</b>10-23DRz can highly block the expression of HBV gene in the 2.2.15 cell model and it can serve as a specific and effective anti-HBV gene therapeutic means.</p>

Cleavage of HCV by HCV specific deoxyribozyme in vitro / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the cleavage activity of specific deoxyribozyme to hepatitis C virus in vitro.</p><p><b>METHODS</b>Three deoxyribozymes were designed to cleave at sites 157, 168, 173 in HCV 5'-noncoding region with the active region of 5'-GGCTAGCTACAACGA-3' respectively. Plasmid pCMV/T7-NCRC -Delta Luc was completely linearized with restriction endonuclease Xba I. HCV RNA5'-NCRC was transcribed in vitro from the linearized products and radiolabelled with [alpha-32P] UTP. Under the conditions of 37 degrees C, pH7.5, Mg2+ 10 mmol/L, the three deoxyribozymes were mixed with substrate RNA individually for 120 minutes and then the reactions were terminated. The cleavaged products were separated with 8% denaturated polyacrylamide gel electrophoresis and displayed by autoradiography. DRz3 was mixed with the substrate RNA at different Mg2+ concentrations. The cleavage efficiency was analyzed with a gel document action analyzing systems.</p><p><b>RESULTS</b>Under the adopted conditions the three deoxyribozymes efficiently cleaved to the target RNA in vitro and the cleavage activity of DRz3 was increased with the increase of Mg2+ concentration.</p><p><b>CONCLUSION</b>The designed deoxyribozymes can cleave 5'-NCR mRNA of HCV efficiently in vitro and it is dose-respondent to Mg2+ concentration.</p>

Inhibition of respiratory syncytial virus replication in cultured cells by RNA-cleaving DNAzyme / 中华儿科杂志

<p><b>OBJECTIVE</b>DNAzyme/Deoxyribozyme is another novel molecular biological tool following the ribozyme. DNAzyme consists of a 15-nucleotide (nt) internal loop as its catalytic domain and two flanking substrate-recognition domains of 7 to 8 nt each which is complementary to substrate. The RNA substrate is cleaved at a particular phosphodiester located between an unpaired purine and a paired pyrimidine residue. DNAzyme has been applied in fields such as viral infectious disease, tumor, cardiovascular disease and genetic disease. But there is no report about using DNAzyme for anti-respiratory syncytial virus purpose. To observe the inhibitory effects of RNA-cleaving DNAzymes on respiratory syncytial virus (RSV) replication in cultured cells.</p><p><b>METHODS</b>Anti-RSV RNA-cleaving DNAzyme DZ604 was designed to target the RSV genome at the start of the NS2 gene in an effort to inhibit the RNA replication. Microscope and electron microscope were used to observe the effects of anti-RSV genomic RNA DNAzyme on cytopathogenic effect (CPE) and ultrastructural change of 9HTE cell induced by RSV infection. Viral plaque forming reduction assay and MTT assay were used to detect the anti-RSV activity and protective function for RSV infected 9HTE cells of DNAzyme.</p><p><b>RESULTS</b>Anti-RSV genomic RNA DNAzyme (DZ604) significantly improved CPE of RSV-infected 9HTE cells. The time to appearance of CPE and of total CPE was delayed by using DZ604 in a dose-dependent manner. At a 5 micro mol/L concentration of DZ604, CPE of 9HTE cells induced by RSV infection at 10 and 1 multiplicity of infection (MOI) was not improved. At smaller MOI (0.1, 0.01, 0.001, 0.0001) of RSV infection, CPE of 9HTE cells was significantly lightened by DZ604 at the same concentration. DZ604 also significantly improved ultrastructural change of 9HTE cells at early stage of RSV infection. Reduction in RSV yield was 85.56% and 8.33% at concentrations of 5 micro mol/L and 0.25 micro mol/L of DZ604. DZ604 inhibited RSV yield in a dose-dependent manner (P < 0.05). Non-specific DNAzyme did not have anti-RSV activity (P > 0.05).</p><p><b>CONCLUSION</b>Anti-RSV genomic RNA DNAzyme designed and synthesized in our laboratory was capable of inhibiting respiratory syncytial virus replication specifically in cultured cells. Our data indicated that DNAzymes could be useful for the prevention against respiratory syncytial virus infection.</p>

Activity of specific deoxyribozymes to cleave hepatitis C virus RNA in vitro / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To analyze the cleavage activity of two deoxyribozymes targeting at hepatitis C virus (HCV) RNA in vitro and evaluate their prospects of antiviral therapy.</p><p><b>METHODS</b>Two specific sequences containing 5' ...A / U... 3' in HCV 5'-noncoding region and 5'-fragment of C region (5'-NCR-C) were selected as the target sites, and with the active region of 5'GGCTAGCTACAACGA3', two phosphorothioate deoxyribozymes (TDRz) named as TDRz-127 and TDRz1 were synthesized. HCV RNA 5'-NCR-C was transcribed in vitro from plasmid pHCV-neo which was completely linearized with restriction endonuclease Nar I, and its 5'-end phosphoric acid was deleted by calf intestinal alkaline phosphatase (CIP), then radiolabelled with T4 polynucleotide kinase and gamma-32P-ATP. Under the conditions such as pH 7.5 and a 10 mmol/L Mg2+ concentration, TDRz-127 and TDRz1 were separately (a 5 micromol/L final concentration) or combinedly (each 2.5 micromol/L) mixed with the substrate RNA (200 nmol/L). After denaturation and then renaturation, the reaction systems were incubated in 37 degrees C, and aliquots were removed to terminate the reaction at intended time points. The cleavage products were separated with 8% denaturated polyacrylamide gel electrophoresis and displayed by autoradiography. Finally, the optical density of each product band was measured with Gel Documentation-Analyzing Systems for calculating the percentages of cleaved HCV 5'-NCR-C.</p><p><b>RESULTS</b>After reaction for 15, 30, 45, 60, 75 and 90 min under the adopted conditions, about 8.3%, 16.1%, 24.3%, 26.2%, 29.4% and 31.1% of HCV 5'-NCR-C was cleaved by TDRz-127 respectively; 7.4%, 13.0%, 15.6%, 18.7%, 19.4% and 20.3% by TDRz1; and 15.1%, 29.6%, 37.8%, 39.1%, 41.5%, 42.6% by combining the two TDRzs.</p><p><b>CONCLUSIONS</b>Cleavage percentage of both TDRz-127 and TDRz1 increases with the time, and the effect of combining the two TDRzs is better than that of anyone.</p>

DNAzymes in vitro inhibit the expression of hepatitis B virus genes / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To evaluate the inhibition effects of DNAzymes specific to Hepatitis B Virus(HBV) s gene and e gene on the expressions of Hepatitis B surface antigen(HBsAg) and Hepatitis B e antigen(HBeAg).</p><p><b>METHODS</b>DNAzymes DrzBS and DrzBC specific to HBV s gene ORF A157UG and e gene ORF A1816UG, respectively, were designed and synthesized. The inhibition effects of DrzBS or DrzBC on the expressions of HBV s and e genes were observed in 2.2.15 cells.</p><p><b>RESULTS</b>The expression of HBV s or e genes was dramatically depressed after 2.2.15 cells treated by DrzBS or DrzBC. The concentration for effective inhibition was within 0.1-2.5 micromol/L and the inhibition showed a dose dependence within that concentration range. The maximum inhibition was 94.2% and 91.8% for DrzBS and DrzBC, respectively. The inhibition was maintained for 72 hours. The efficiency of inhibiting HbsAg, HbeAg in 2.2.15 cells by DrzBS, DrzBC was higher than that by antisense oligonucleotides for the same target genes. The concentrations for effective inhibition of the DNAzymes were at least 10-fold lower compared with antisense oligonucleotides. Neither inhibition on the replication of HBV DNA nor toxicity to 2.2.15 cells was observed.</p><p><b>CONCLUSION</b>DrzBS and DrzBC can highly block the expressions of HBV s gene and e gene in 2.2.15 HBV cell model and are proved a specific and effective anti-HBV gene therapeutic means.</p>