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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.

Objective: To investigate the cleavage activities of 10-23 DNA enzymes targeting at HBV C gene mRNA in vitro., Methods: 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., Result: 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., Conclusion: 10-23 DNA enzymes targeting at HBV C gene mRNA possess the specific cleavage activities in vitro.

Objective: To study the cleavage of the deoxyribozyme targeting Period1 (Per1) mRNA in vitro and its effect on the opiate-induced reward in mice., Method: The specific deoxyribozyme (DRz164) targeting Per1 mRNA was designed after MFold analysis and being synthesized chemically. The cleavage reactions containing DRz164 and Per1 RNA fragments transcripted in vitro were performed under certain conditions. DIG Nucleic Acid Detection Kit was used to detect the cleavage activity of DRz164. The morphine-induced reward in mice was observed in a conditioned place preference test after pretreatment with DRz164., Result: When DRz164 and Per1 RNA transcripts were incubated under certain conditions with 37 degrees C for 30, 60, 90, and 120 min, about 36.4%, 40.5%, 47.8%, 63% of Per1 RNA transcripts were cleaved by DRz164 respectively. After pretreated with the deoxyribozyme, mice didn't show a morphine-induced place preference., Conclusion: DRz164 targeting per1 mRNA has the specific cleavage activity which increases with the time in vitro. Suppression of morphine-induced place preference occurs by pretreatment with the deoxyribozyme. Mice show a lack of morphine-induced reward, which alleviates the psychic dependence on the morphine.

Locked nucleic acid (LNA) is a novel oligonucleotide analogue in which 2'-O and 4'-C positions in the b-D-ribofuranosyl ring are joined via an O-methylene, S-methylene or amino-methylene moiety, locked in a C3'-endol3E north (N)-type furanose conformation. LNAs posses many properties, such as extraordinarily high hybridize affinities for complementary DNA/RNA sequences, remarkable antisense activity, nuclease resistance, good aqueous solubility and none detactable toxicity, et al. LNAs is a most promising molecule for development of diagnostics and therapeutics. For example, apply LNAs to single--nucleotide polymorphisms genotyping, telomerase activity have been efficiently suppressed by LNA oligomers, efficient cleavage of highly structured RNA has been achieved using LNA-modified DNAzymes (LNAzymes), and so on.

Objective: To analyze the cleavage activity of two deoxyribozymes targeting at hepatitis C virus (HCV) RNA in vitro and evaluate their prospects of antiviral therapy., Methods: 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., Results: 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., Conclusions: 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.