title: Inhibition of DNA replication of human papillomavirus by using zinc finger-single-chain FokI dimer hybrid.
abstract: Previously, we reported that an artificial zinc-finger protein (AZP)-staphylococcal nuclease (SNase) hybrid (designated AZP-SNase) inhibited DNA replication of human papillomavirus type 18 (HPV-18) in mammalian cells by binding to and cleaving a specific HPV-18 ori plasmid. Although the AZP-SNase did not show any side effects under the experimental conditions, the SNase is potentially able to cleave RNA as well as DNA. In the present study, to make AZP hybrid nucleases that cleave only viral DNA, we switched the SNase moiety in the AZP-SNase to the single-chain FokI dimer (scFokI) that we had developed previously. We demonstrated that transfection with a plasmid expressing the resulting hybrid nuclease (designated AZP-scFokI) inhibited HPV-18 DNA replication in transient replication assays using mammalian cells more efficiently than AZP-SNase. Then, by linker-mediated PCR analysis, we confirmed that AZP-scFokI cleaved an HPV-18 ori plasmid around its binding site in mammalian cells. Finally, a modified MTT assay revealed that AZP-scFokI did not show any significant cytotoxicity. Thus, the newly developed AZP-scFokI hybrid is expected to serve as a novel antiviral reagent for the neutralization of human DNA viruses with less fewer potential side effects.
authors.affiliation: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyotodaigaku-Katsura, Nishikyo-ku, Kyoto, 615-8510, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Mino
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Mori
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Aoyama
authors.forename: Takashi
authors.initials: T
authors.lastname: Sera
mesh_terms: D001665:Binding Sites / Q000235:genetics; D001709:Biotechnology; D004261:DNA Replication / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000633:toxicity; D005260:Female; D057809:HEK293 Cells; D052161:Human papillomavirus 18 / Q000235:genetics* / Q000378:metabolism* / Q000472:pathogenicity; D006801:Humans; D008836:Micrococcal Nuclease / Q000235:genetics / Q000633:toxicity; D016133:Polymerase Chain Reaction; D013379:Substrate Specificity; D014162:Transfection; D016335:Zinc Fingers / Q000235:genetics
chemical_list: D004279:DNA, Viral; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific; D008836:Micrococcal Nuclease