Abstract
Zinc-finger-FokI nucleases (ZFNs) are useful for manipulating genomic DNA, but two ZFNs are required to cleave one site of double-stranded DNA (dsDNA), which limits the choice of targets. To refine ZFN technology, we constructed artificial zinc-finger nucleases containing an artificial zinc-finger protein (AZP) and a single-chain FokI dimer with nine different peptide linkers between two FokI molecules (designated AZP-scFokI). DNA cleavage assays revealed that the AZP-scFokI variant possessing the longest peptide linker cleaved dsDNA with equal or greater reactivity than the corresponding AZP-FokI dimer. The DNA cleavage pattern of AZP-scFokI suggests that the enhanced dsDNA cleavage was due to increased formation of FokI dimer in AZP-scFokI. Furthermore, we demonstrated that AZP-scFokI site-specifically cleaved its target DNA due to the AZP moiety discriminating one base pair difference. Thus, a single AZP-scFokI molecule is able to cleave dsDNA efficiently and site-specifically, and enhances the usefulness of the ZFN approach.
| Original language | English |
|---|---|
| Pages (from-to) | 156-161 |
| Number of pages | 6 |
| Journal | Journal of Biotechnology |
| Volume | 140 |
| Issue number | 3-4 |
| DOIs | |
| Publication status | Published - Mar 25 2009 |
| Externally published | Yes |
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Keywords
- Artificial zinc-finger protein
- DNA cleavage
- Protein engineering
- Zinc-finger nuclease
ASJC Scopus subject areas
- Biotechnology
- Applied Microbiology and Biotechnology
Cite this
Efficient double-stranded DNA cleavage by artificial zinc-finger nucleases composed of one zinc-finger protein and a single-chain FokI dimer. / Mino, Takashi; Aoyama, Yasuhiro; Sera, Takashi.
In: Journal of Biotechnology, Vol. 140, No. 3-4, 25.03.2009, p. 156-161.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Efficient double-stranded DNA cleavage by artificial zinc-finger nucleases composed of one zinc-finger protein and a single-chain FokI dimer
AU - Mino, Takashi
AU - Aoyama, Yasuhiro
AU - Sera, Takashi
PY - 2009/3/25
Y1 - 2009/3/25
N2 - Zinc-finger-FokI nucleases (ZFNs) are useful for manipulating genomic DNA, but two ZFNs are required to cleave one site of double-stranded DNA (dsDNA), which limits the choice of targets. To refine ZFN technology, we constructed artificial zinc-finger nucleases containing an artificial zinc-finger protein (AZP) and a single-chain FokI dimer with nine different peptide linkers between two FokI molecules (designated AZP-scFokI). DNA cleavage assays revealed that the AZP-scFokI variant possessing the longest peptide linker cleaved dsDNA with equal or greater reactivity than the corresponding AZP-FokI dimer. The DNA cleavage pattern of AZP-scFokI suggests that the enhanced dsDNA cleavage was due to increased formation of FokI dimer in AZP-scFokI. Furthermore, we demonstrated that AZP-scFokI site-specifically cleaved its target DNA due to the AZP moiety discriminating one base pair difference. Thus, a single AZP-scFokI molecule is able to cleave dsDNA efficiently and site-specifically, and enhances the usefulness of the ZFN approach.
AB - Zinc-finger-FokI nucleases (ZFNs) are useful for manipulating genomic DNA, but two ZFNs are required to cleave one site of double-stranded DNA (dsDNA), which limits the choice of targets. To refine ZFN technology, we constructed artificial zinc-finger nucleases containing an artificial zinc-finger protein (AZP) and a single-chain FokI dimer with nine different peptide linkers between two FokI molecules (designated AZP-scFokI). DNA cleavage assays revealed that the AZP-scFokI variant possessing the longest peptide linker cleaved dsDNA with equal or greater reactivity than the corresponding AZP-FokI dimer. The DNA cleavage pattern of AZP-scFokI suggests that the enhanced dsDNA cleavage was due to increased formation of FokI dimer in AZP-scFokI. Furthermore, we demonstrated that AZP-scFokI site-specifically cleaved its target DNA due to the AZP moiety discriminating one base pair difference. Thus, a single AZP-scFokI molecule is able to cleave dsDNA efficiently and site-specifically, and enhances the usefulness of the ZFN approach.
KW - Artificial zinc-finger protein
KW - DNA cleavage
KW - Protein engineering
KW - Zinc-finger nuclease
UR - http://www.scopus.com/inward/record.url?scp=62849090902&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=62849090902&partnerID=8YFLogxK
U2 - 10.1016/j.jbiotec.2009.02.004
DO - 10.1016/j.jbiotec.2009.02.004
M3 - Article
VL - 140
SP - 156
EP - 161
JO - Journal of Biotechnology
JF - Journal of Biotechnology
SN - 0168-1656
IS - 3-4
ER -