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deoxyribozymes inhibit the expression of periodl gene in vitro

Science China(Life Sciences) 2005年第3期48卷 195-201页

作者： ZHOU Wei WANG Yueqi LIU Yanyou PENG Wenzhen XIAO Jing ZHU Bin WANG Zhengrong Biomedical Engineering Department School of West China Basic and Forensic ScienceSichuan UniversityChengdu610041China Molecular Biology and Biology Chemistry Department School of West China Basic and Forensic ScienceSichuan UniversityChengdu610041China

To investigate the effect of two deoxyribozymes targeting period1(per1)mRNA in vitro for exploring a novel gene therapy approach about circadian rhythm diseases,the specific deoxyribozymes targeting per1 were designed and synthesized chemically following MFold analysis according to its mRNA secondary ***1 RNA fragments were prepared by in vitro transcription of pcDNA3.1(+)-per1_(164:256).The cleavage reactions containing deoxyribozymes and per1 RNA fragments were performed under certain *** the transfection tech-nique mediated by LipofectAMINE^(TM),pcDNA3-per1 and DRz164 or DRz256 were introduced into NIH3T3 *** effects of deoxyribozymes on per1 were studied by reverse tran-script-polymerase chain reaction(RT-PCR)and flow cytometry(FCM).When deoxyribozymes and RNA transcripts were incubated under the adopted conditions at 37℃for 2 h,about 63%of per1_(164:256)RNA transcripts were cleaved by DRz164 and about 50.5%by *** co-transfecting pcDNA3-per1 with DRz164 or DRz256,the expression of per1 mRNA was de-creased,as indicated by RT-PCR semi-quantity *** analysis showed that Per1 protein was *** DRz164 and DRz256 targeting per1 have the specific cleavage activity to-ward per1 mRNA in vitro and can highly block the expression of per1 gene in cellular milieu.

关键词： period1 gene deoxyribozymes cleavage in vitro gene transfection.

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Design and activity evaluation of deoxyribozymes specifically targeting hepatitis C virus RNA

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Journal of Medical Colleges of PLA(China) 2003年第3期18卷 143-149页

作者：于乐成王宇明王升启顾长海毛青陈忠斌刘鸿凌 Institute of Infectious Diseases of Chinese PLA Southwest Hospital Third Military Medical University Chongqing 400038 China Institute of Radiation Medicine Academy of Military Medical Science Beijing 100850 China

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-transfe

关键词： deoxyribozymes hepatitis C virus in vitro transcription transgeneic therapy

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The Possibility of Nucleic Acids to Act as Anti-Viral Therapeutic Agents—A Review

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Open Journal of Medical Microbiology 2021年第3期11卷 198-248页

作者： Md. Selim Reza Farzana Mim Mohammad Rezaul Quader Mohammad Jahidur Rahman Khan Md. Sabir Hossain Kazi Rasel Uddin Salina Akter Sharmin Rahman Sumon Roy Md. Abdullah Rumman RT-PCR LAB Bangabandhu Sheikh Mujib Medical College Faridpur Bangladesh Department of Biochemistry and Molecular Biology Jahangirnagar University Dhaka Bangladesh Department of Biochemistry Bangabandhu Sheikh Mujib Medical College Faridpur Bangladesh Department of Microbiology Shaheed Suhrawardy Medical College Dhaka Bangladesh Department of Pharmacology Ibrahim Medical College Dhaka Bangladesh

For just about 30 years, researchers have considered the likelihood to utilize nucleic acids as antiviral therapeutics. In principle, small single-stranded nucleotide sequence (oligonucleotide) could hybridize to a particular gene or messenger RNA and diminish transcription or translation, respectively, in this manner decreasing the amount of protein that is synthesized. Until now, an incredible number of antisense oligonucleotides, double-stranded oligonucleotides, aptamers, ribozymes, deoxyribozymes, interfering RNAs, chimeric RNA-DNA molecules, antibody genes has been created artificially and applied effectively for comprehension and manipulating biological processes and in clinical preliminaries to treat a variety of diseases. Their versatility and potency make them similarly fit candidates for fighting viral infections. However, troubles with their efficiency, off-target effects, toxicity, delivery, and stability halted the development of nucleic acid-based therapeutics that can be utilized in the clinic. The potential for nucleic acid therapeutic agents is significant and is quite recently beginning to be realized. In this review, we have summarized some of the recent advancements made in the area of nucleic acid based therapeutics and focused on the methods of their delivery and associated challenges.

关键词： Antisense Oligonucleotides Aptamers Ribozymes deoxyribozymes RNA Interference

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