| Human Respiratory syncytial virus (hRSV) is the leading cause of acute upper and lower respiratory tract infections in infants, young children, and adults worldwide. Although several efforts are underway, neither a reliable vaccine nor a specific anti-RSV agent is currently available. Deoxyribozyme (DNAzyme, DZ) is a novel approach which is being developed to specifically cleave RNA molecules at almost any targeted sequence. Based on RNA-cleaving function, DZ is derived from a combinatorial library of sequences by in vitro selection. In our previous studies, we have already developed two DZs, DZ604 and DZ8269, against RSV A2 strain. Their anti-RSV activities were demonstrated both in culture cells and in RSV infectious mouse model. To develop a universal DZ and test its antiviral activity against both genotypes of RSV is our major goal in the present study.PART I Selection of Binding Sites and Design of Specific DZ against Both Subgroups of RSVThree main basic factors should be considered for DZ cleavage site selection against the target RNA. Firstly, the target site should be vital for the virus replication. Secondly, target sequence should be highly conserved among different strains of RSV. Thirdly, predicted by the computer-assisted analysis of the secondary structure of RNA, the target site should be easy to assess and located at an unstable position. According to the three basic rules in target site choosing, in the present study, we designed two RNA cleaving 10-23 DZs, DZ1133 and DZ7601. They were designed to target the transcription start signal for N gene and M2 gene of RSV genomic RNA respectively. As a control, two 19nt antisense oligonucleotides targeted the same substrate (AS1133 and AS7601) and two mutant DZs (mutDZ1133 and mutDZ7601) were introduced in the tests. In order to increase the stability of the DZ and the AS, the first two bases at the 5' end as well as the last three bases at the 3' end were substituted with phosphorothioates. In addition to phosphorothioate modification, these oligonucleotides used in the cell culture experiments were conjugated with cholesterol at the 3' end.PART II Cleavage Effect of DZ on RSV RNA in Cell Free System79Background and Objective We employed two 10-23 DZs, DZ1133 and DZ7601, which were expected to cut the start signal of RSV N and M2 genoraic RNA. In this part, their catalytic activities were analyzed by cell free assays. The time- and ion- dependent cleavage capacity of DZ were investigated as well.Methods 403bp cDNA of N gene and 402bp cDNA of M2 gene from CHI8537 strain were generated by RT-PCR. The amplified fragments were inserted into pBluescript II KS+, generated two combinated phagemids, pN-18537 and pM2-18537. The RNA fragments of N and M2 were transcribed in vitro using T7 polymerase. The RNA cleavage activities of DZ, mutDZ, and AS were measured in cell free system.Results DZ1133 was capable of cutting RSV N genome RNA at the expected site in sequence- and time-dependent manner. The catalytic activity of DZ1133 was increasing along with the rising of Mg2+ concentration from 10 mM to 25 mM, but no cleavage was observed when the reaction was performed under simulated physiological intracellular conditions of Mg2+ (2mM). There was no any detectable cleavage activity of DZ7601. Both AS and mutDZ failed to cleave their substrate RNA.Conclusion DZ1133 designed toward RSV N genome RNA can cleavage the substrate RNA specifically and efficiently in the cell free system. This catalytic activity is time- and Mg2+-dependent. DZ7601 expected to cleave the RSV M2 genome RNA failed to show any cleavage activity. MutDZ1133 as well as AS1133 also lack of cleavage activity to80PART III Isolation, Culture, and Identification of Local Strainsof RSV in ChongqingBackground and Objective In order to develop a universal DZ and demonstrate its antiviral activity against both genotypes of RSV, local strains of RSV in Chongqing were isolated, cultured, identified and tittered. Their subgroups were determined as well.Methods Nasopharyn...
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