title: DNA aptamers against FokI nuclease domain for genome editing applications.
abstract: Genome editing with site-specific nucleases (SSNs) can modify only the target gene and may be effective for gene therapy. The main limitation of genome editing for clinical use is off-target effects; excess SSNs in the cells and their longevity can contribute to off-target effects. Therefore, a controlled delivery system for SSNs is necessary. FokI nuclease domain (FokI) is a common DNA cleavage domain in zinc finger nuclease (ZFN) and transcription activator-like effector nuclease. Previously, we reported a zinc finger protein delivery system that combined aptamer-fused, double-strand oligonucleotides and nanoneedles. Here, we report the development of DNA aptamers that bind to the target molecules, with high affinity and specificity to the FokI. DNA aptamers were selected in six rounds of systematic evolution of ligands by exponential enrichment. Aptamers F6#8 and #71, which showed high binding affinity to FokI (Kd=82nM, 74nM each), showed resistance to nuclease activity itself and did not inhibit nuclease activity. We immobilized the ZFN-fused GFP to nanoneedles through these aptamers and inserted the nanoneedles into HEK293 cells. We observed the release of ZFN-fused GFP from the nanoneedles in the presence of cells. Therefore, these aptamers are useful for genome editing applications such as controlled delivery of SSNs.
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan.
authors.forename: Maui
authors.initials: M
authors.lastname: Nishio
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Matsumoto
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 5 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan.
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Kato
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan.
authors.forename: Koichi
authors.initials: K
authors.lastname: Abe
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan.
authors.forename: Jinhee
authors.initials: J
authors.lastname: Lee
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan.
authors.forename: Kaori
authors.initials: K
authors.lastname: Tsukakoshi
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 5 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan.
authors.forename: Ayana
authors.initials: A
authors.lastname: Yamagishi
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan; Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 5 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan.
authors.forename: Chikashi
authors.initials: C
authors.lastname: Nakamura
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan. Electronic address: ikebu@cc.tuat.ac.jp.
authors.forename: Kazunori
authors.initials: K
authors.lastname: Ikebukuro
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry*; D015374:Biosensing Techniques; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000302:isolation & purification*; D000072669:Gene Editing; D005818:Genetic Engineering; D016678:Genome; D057809:HEK293 Cells; D006801:Humans; D016335:Zinc Fingers / Q000235:genetics
chemical_list: D052157:Aptamers, Nucleotide; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific
keywords: Aptamer; FokI; Genome editing; Protein delivery