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 desig...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(+)-per1164:256. The cleavage reactions containing deoxyribozymes and per1 RNA fragments were performed under certain conditions. With the transfection tech- nique mediated by LipofectAMINETM, 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 per1164:256 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.展开更多
<span style="font-family:""><span style="font-family:Verdana;">For just about 30 years, researchers have considered the likelihood to utilize </span><span style="font...<span style="font-family:""><span style="font-family:Verdana;">For just about 30 years, researchers have considered the likelihood to utilize </span><span style="font-family:Verdana;">nucleic acids as antiviral therapeutics. In principle, small single-stranded</span><span style="font-family:Verdana;"> nuc</span><span style="font-family:Verdana;">leotide sequence (oligonucleotide) could hybridize to a particular gene or</span><span style="font-family:Verdana;"> mes</span><span style="font-family:Verdana;">senger RNA and diminish transcription or translation, respectively, in this</span><span style="font-family:Verdana;"> 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</span></span><span style="font-family:Verdana;">-</span><span style="font-family:""><span style="font-family:Verdana;">DNA molecules, antibody genes has been created artificially and ap</span><span style="font-family:Verdana;">plied effectively for comprehension and manipulating biological processe</span><span style="font-family:Verdana;">s 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.展开更多
基金This work was supported by the National Natural Science Foundation of China(Grant Nos.3007027&39970275)
文摘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(+)-per1164:256. The cleavage reactions containing deoxyribozymes and per1 RNA fragments were performed under certain conditions. With the transfection tech- nique mediated by LipofectAMINETM, 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 per1164:256 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.
文摘<span style="font-family:""><span style="font-family:Verdana;">For just about 30 years, researchers have considered the likelihood to utilize </span><span style="font-family:Verdana;">nucleic acids as antiviral therapeutics. In principle, small single-stranded</span><span style="font-family:Verdana;"> nuc</span><span style="font-family:Verdana;">leotide sequence (oligonucleotide) could hybridize to a particular gene or</span><span style="font-family:Verdana;"> mes</span><span style="font-family:Verdana;">senger RNA and diminish transcription or translation, respectively, in this</span><span style="font-family:Verdana;"> 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</span></span><span style="font-family:Verdana;">-</span><span style="font-family:""><span style="font-family:Verdana;">DNA molecules, antibody genes has been created artificially and ap</span><span style="font-family:Verdana;">plied effectively for comprehension and manipulating biological processe</span><span style="font-family:Verdana;">s 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.