title: Sandwiched zinc-finger nucleases harboring a single-chain FokI dimer as a DNA-cleavage domain.
abstract: Zinc-finger nucleases (ZFNs) are a powerful tool for manipulation of genomic DNA. Recently, we reported a new ZFN composed of one artificial zinc-finger protein (AZP) and a single-chain FokI dimer (scFokI) that refines ZFN technology. While AZP-scFokI cleaved DNA specifically around the AZP-target site, several nucleotide positions were cleaved due to the mobility of the scFokI domain. In the present study, we aimed to improve the DNA-cleavage specificity at the nucleotide level. To this end, we sandwiched a scFokI domain between two AZPs to reduce the mobility of the scFokI moiety when bound to DNA. We demonstrated that the AZP-sandwiched scFokI cleaved DNA at a single nucleotide position of a target plasmid, in which two AZP-binding sites were connected with a 6-bp spacer, with multiple turnovers. Further improvement of AZP-sandwiched scFokI will lead to development of ideal artificial meganucleases.
authors.affiliation: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyotodaigaku-Katsura, Kyoto 615-8510, Japan.
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Mori
authors.forename: Ikuko
authors.initials: I
authors.lastname: Kagatsume
authors.forename: Kazuki
authors.initials: K
authors.lastname: Shinomiya
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Aoyama
authors.forename: Takashi
authors.initials: T
authors.lastname: Sera
mesh_terms: D004247:DNA / Q000737:chemistry*; D053837:DNA Cleavage; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000235:genetics; D015202:Protein Engineering; D055503:Protein Multimerization; D016335:Zinc Fingers
chemical_list: D004247:DNA; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific