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

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.

Deoxyribozymes inhibit the expression of periodl gene in vitro

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 structure.per1 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 conditions.With the transfection tech-nique mediated by LipofectAMINE^(TM),pcDNA3-per1 and DRz164 or DRz256 were introduced into NIH3T3 cells.The 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 DRz256.After co-transfecting pcDNA3-per1 with DRz164 or DRz256,the expression of per1 mRNA was de-creased,as indicated by RT-PCR semi-quantity analysis.FCM analysis showed that Per1 protein was inhibited.Both 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.

Ribozyme是唯一的非酶生物催化剂吗?

人们一直认为酶是唯一的生物催化剂，酶和生物催化剂概念具有等价性。Ｒｉｂｏｚｙｍｅ的发现打破了这种等价性，生物催化剂上升为属概念，酶和ｒｉｂｏｚｙｍｅ并列成为种概念。随着研究的深入，人们发现ＤＮＡ也具有催化活性。本实验室对博莱霉素（ＢＬＭ）的研究表明，ＢＬＭ介导的ＤＮＡ断链反应具有许多类似酶促反应的特性，并推测ＢＬＭ和生物体内的许多辅酶可能是生物催化分子进化过程中保留下来的古老而简单的小分子生物催化剂。可以断言ｒｉｂｏｚｙｍｅ并非唯一的非酶生物催化剂。

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

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.

酶性DNA(deoxyribozyme)研究进展

Deoxyribozyme(酶性 DNA)自发现以来 ,正由基础研究开始走向应用。本文综述了 deoxyribozyme研究的发展过程 ,并探讨了 deoxyribozym