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To investigate the effect of two deoxyribozymes targeting periodl (perl) mRNA in vitro for exploring a novel gene therapy approach about circadian rhythm diseases, the specific deoxyribozymes targeting perl were designed and synthesized chemically following MFold analysis according to its mRNA secondary structure, perl RNA fragments were prepared by in vitro transcription of pcDNA3.1 (+)-perl164:256. The cleavage reactions containing deoxyribozymes and perl RNA fragments were performed under certain conditions. With the transfection technique mediated by LipofectAMINETM, pcDNA3-perl and DRz164 or DRz256 were introduced into NIH3T3 cells. The effects of deoxyribozymes on perl 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℃ for 2 h, about 63% of perl164:256 RNA transcripts were cleaved by DRz164 and about 50.5% by DRz256. After cotransfecting pcDNA3-perl with DRz164 or DRz256, the expression of perl mRNA was decreased, as indicated by RT-PCR semi-quantity analysis. FCM analysis showed that Perl protein was inhibited. Both DRz164 and DRz256 targeting perl have the specific cleavage activity toward perl mRNA in vitro and can highly block the expression of perl gene in cellular milieu.