Deoxyribozymes inhibit the expression ofperiod1 genein vitro

To investigate the effect of two deoxyribozymes targetingperiod1 (per1) mRNAin vitro for exploring a novel gene therapy approach about circadian rhythm diseases, the specific deoxyribozymes targetingper1 were designed and synthesized chemically following MFold analysis according to its mRNA secondary structure.per1 RNA fragments were prepared byin vitro transcription of pcDNA3.1(+)-per1 164:256. The cleavage reactions containing deoxyribozymes andper1 RNA fragments were performed under certain conditions. With the transfection technique mediated by Lipofect AMINE™, pcDNA3-per1 and DRz164 or DRz256 were introduced into NIH3T3 cells. The effects of deoxyribozymes onper1 were studied by reverse transcript-polymerase chain reaction (RT-PCR) and flow cytometry (FCM). When deoxyribozymes and RNA transcripts were incubated under the adopted conditions at 37°C for 2 h, about 63% ofper1 164:256 RNA transcripts were cleaved by DRz164 and about 50.5% by DRz256. After cotransfecting pcDNA3-per1 with DRz164 or DRz256, the expression ofper1 mRNA was decreased, as indicated by RT-PCR semi-quantity analysis. FCM analysis showed that Per1 protein was inhibited. Both DRz164 and DRz256 targetingper1 have the specific cleavage activity towardper1 mRNAin vitro and can highly block the expression ofper1 gene in cellular milieu.