Nucleic Acids Research Advance Access originally published online on May 29, 2008
Nucleic Acids Research 2008 36(12):3926-3938; doi:10.1093/nar/gkn313
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Nucleic Acids Research, 2008, Vol. 36, No. 12 3926-3938
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
Development of a single-chain, quasi-dimeric zinc-finger nuclease for the selective degradation of mutated human mitochondrial DNA
1MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, 2MRC Dunn Human Nutrition Unit, Hills Road, Cambridge CB2 0XY, UK and 3Sangamo BioSciences, Inc., 501 Canal Boulevard, Suite A100, Richmond, CA 94804, USA
*To whom correspondence should be addressed. Tel: +44 1223 252843; Fax: +44 1223 252900; Email: michal.minczuk{at}mrc-dunn.cam.ac.uk
Received March 28, 2008. Revised May 1, 2008. Accepted May 1, 2008.
The selective degradation of mutated mitochondrial DNA (mtDNA) molecules is a potential strategy to re-populate cells with wild-type (wt) mtDNA molecules and thereby alleviate the defective mitochondrial function that underlies mtDNA diseases. Zinc finger nucleases (ZFNs), which are nucleases conjugated to a zinc-finger peptide (ZFP) engineered to bind a specific DNA sequence, could be useful for the selective degradation of particular mtDNA sequences. Typically, pairs of complementary ZFNs are used that heterodimerize on the target DNA sequence; however, conventional ZFNs were ineffective in our system. To overcome this, we created single-chain ZFNs by conjugating two FokI nuclease domains, connected by a flexible linker, to a ZFP with an N-terminal mitochondrial targeting sequence. Here we show that these ZFNs are efficiently transported into mitochondria in cells and bind mtDNA in a sequence-specific manner discriminating between two 12-bp long sequences that differ by a single base pair. Due to their selective binding they cleave dsDNA at predicted sites adjacent to the mutation. When expressed in heteroplasmic cells containing a mixture of mutated and wt mtDNA these ZFNs selectively degrade mutated mtDNA, thereby increasing the proportion of wt mtDNA molecules in the cell. Therefore, mitochondria-targeted single-chain ZFNs are a promising candidate approach for the treatment of mtDNA diseases.
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