Tnhibition Of Influenza A Virus Replication By M2 Gene And PB1 Gene Targered Dnazyme In Vitro And Viro

PART ONE: SELECTION OF BINDING SITES AND DESIGN,MODIFICATION AND SYNTHESIS OF SPECIFIC DNA ENZYME AGAINST IFAVObjective To design and modification of DNA zyme target influenza A virusMethods Three main basic factors should be considered for the selection of DNAzyme cleavage site against the target RNA. Firstly, the target site should be essential for the virus transcription and replication. Secondly, target sequence should be highly conserved among different strains of IFAV. Thirdly, predicted by computer-assisted analysis of the secondary structure of IFAV, the target site should be easy to assess and located at an unstable position.Results According to the three basic rules in target site choosing, in the present study, we designed two RNA cleaving 10-23DZs-DZ-M2 and DZ-PB1. With the computer-assisted analysis of the secondary structure of RNA, the target of M2 and PB1 gene signal should be unstably paired and accessible to DZs. They were designed to target the transcription start signal site -AUG for M2 and PB1 gene of IFAV mRNA respectively.As a control, two 19nt antisense oligonucleotides target the same substrate(ASODN-M2 and ASODN-PB1) and two mutant DZs (mutDZ-M2 and mutDZ-PB1) were introduced in the tests. In order to increase the stability of DZs and ASODN the first three bases at the 5'endas well as the last three bases at the 3'end were substituted with phosphorothioates. In addition to phosphorothioate modification ,these oligonucleotides used ini the cell culture experiments were transfected by Lipofectamine TM2000. All DZs and antisense oligonucleotides were conjugated with fluorescent (5′-FITC). Oligonucleotides sequences were chemically synthesized from Invitrogen?.The oligonucleotides sequences as follows : Conclusion This successful designed and modified DNA enzyme (DZ-M2,DZ-PB1) and control antisense oligonucleotide (ASODN-M2,ASODN-PB1 ),mutDZ-M2 and mutDZ-PB1 for following experiments.PART TWO: THE OBTAIN OF THE RNA TEMPLATE FOR CLEAVAGE EFFECT OF DZ IN CELL FREE SYSTERMObjective To obtain the RNA template for observing cleavage effect of DZ in cell free system.Methods 304bp cDNAs of M2 gene and 253bp cDNAs of PB1 gene were generated by RT-PCR from mRNA of IFAV (H1N1) using specific primers, and were inserted into the plasmid of pBluescript II KS+ (Stratagene, La Jolla, CA) vector containing T7 promotor before in vitro transcription with T7 polymerase using MAXIscript kit(Ambion,Austin,TX,USA). These recombinant pasmids were transformed into JM109 strain. Finally the recombinant plasmids PBKS-M2,PBKS-PB1 were confirmed by restriction enzyme mapping,colony PCR and DNA sequencing. The RNA fragment of M2 and PB1 gene was transcribed in vitro using T7 polymerase and 1ug PBKS-M2 or PBKS-PB1 digested with HindIII by MAXIscript kit.The RNA cleavage activities of DZ,mutDZ,ASODN were measured in cell free system.Results The recombinant plasmids(PBKS-M2 and PBKS-PB1) targeting the mRNA of IFAV M2 and PB1 gene were successfully constructed and confirmed by DNA sequencing. The RNA fragments of M2 and PB1 were transcribed in vitro using T7 ploymerase.Conclusion Two recombinant plasmids PBKS-M2 and PBKS-PB1 targeting the M2 gene and PB1 gene of IFAV were successfully constructed and the RNA fragment of M2 and PB1 gene were transcribed in vitro using T7 polymerase .PART THREE: CLEAVAGE EFFECT OF DZ ON IFAV RNA IN CELL FREE SYSTERMObjective To explore the DNA enzyme cleavage activity of DNA enzymes on the target sequence of IFAV mRNA in cell free systerm. Methods To assess the cleavage effect of DNAzymes to the target sequence of IFAV mRNA , The RNA cleavage activities of DZ,ASODN and mutDZ were performed in cell free system.. One picomole transcript RNA was subjected to in vitro cleavage with 1 pmol DZ (or ASODN,mutDZ) by incubation at 37℃in 10mM MgCl2, 250mM Tris–HCl (pH 7.5) solution for 60, 90, 120,180 and 240 min. The cleavage products were electrophorized on 8% polyacrylamide–7M urea gels, and quantified with a densitometer (Totallab, Nonlinear Dynamics Ltd., UK) after silver stain according to directions. The transfection efficiency of LipofectamineTM2000 was tested by flow cytometric analysis. MTT assay was conducted for testing the cytotoxicity of DZ transfected by LipofectamineTM2000 in cultured MDCK cells.Results One of DNAzymes, DZ-M2, targeted against IFAV mRNA, was found to be most efficient in the cleavage of 304 nt in vitro transcribed M2 gene RNA substrate at the anticipated position after 2 h incubation, generating the size-matching products with 114 and 189 nt fragments. Moreover, RNA substrates decreased and the products increased with the incubation time prolonged in the cleavage reaction, resulting the cleavage percentages of 30.85%, 38.50%, 58.90%, 60.85% and 68.90% at the incubation time of 60, 90, 120, 180 and 240 min, respectively. There were same cleavage effection detectable at DZ-PB1. There was no any detectable cleavage activity of mutDZ nor 21 nt antisense oligonucleotide (ASODN) targeting the same sequences gave any detectable cleaved products , suggesting the sequence-specific, time-dependent and catalytic cleavage capacity of DZ on IFV mRNA. Therefore, we used DZ-M2,DZ-PB1for further investigations. The transfection efficiency of LipofectamineTM2000 (99.82%) was higher than DZ only modified with phosphorothioates(1.3%) by flow cytometric analysis. The apparent toxicity of DZ transfected by LipofectamineTM2000 into MDCK cells were not observed.Conclusion DZ-M2,DZ-PB1 designed toward IFAV M2 and PB1 m RNA can cleavage the substrate RNA specifically and efficiently in the cell free system.This catalytic activity is time–dependent. MutDZ and ASODN lack of cleavage activity to their RNA substrate.PART FOUR: INHIBITORY ACTIVITY OF DZ-M2 AND DZ-PB1 ON IFAV REPLICATION IN CULTURED CELLSObjective The catalytic ability of active DZ–M2,DZ-PB1 and its control ASODN were tested in cultured IFAV infected cells.Methods Effect of DZs and ASODNs on the CPE caused by IFAV was observed microscopically and measured by quantitative colorimetric MTT assay. MTT assay was conducted for testing the cell survival rate of these oligonucleotides in cultured infected MDCK cells as well.Virus yield reduction caused by these oligonucleotides was tittered by plaque forming assay in MDCK cells. RT-PCR,Westernblot technique and immunofluorescence assay were performed for evaluating the inhibitory activity of DZ-M2 and DZ-PB1 on RNA expression and protein expression respectively. Result DZ-M2,DZ-PB1 blocked the CPE caused by IFAV in a dose–dependent manner.Both DZ-M2 and DZ-PB1appeared to inhibit IFAV in lower concentration (0.3μm) than ASODN-M2 and ASODN-PB1 and showed a dose–dependent. Both DZ and ASODN showed reduction in IFAV yields and DZ showed a more potent inhibition activity than ASODN. DZ was capable of decressing the IFAV mRNA expression and proteins expression.Conclusion DZ-M2,DZ-PB1 which targeted the mRNA of IFAV gene showed a significant and specific inhibitory effect upon IFAV replication both in mRNA and protein level in in cells culture system.Since it is more active than ASODN-M2,ASODN-PB1,DZ-M2,DZ-PB1 might be a valid alternative or in combination with the present treatment of IFAV infection. These study indicate that the PB1 and M2 mRNA may play an important role in regulating IFAV replication and ASODN may have inhibitory activity on IFAV replication.Further studies in vivo will be needed to determine whether DZ-M2,DZ-PB1 could provide benefits to IFAV and H5N1 treatment. The resuIts established the basis for further study on new drugs against IFAV infection include highly pathogenic avian influenza virus...