Development of a single-chain, quasi-dimeric zinc-finger nuclease for the selective degradation of mutated human mitochondrial DNA.

TitleDevelopment of a single-chain, quasi-dimeric zinc-finger nuclease for the selective degradation of mutated human mitochondrial DNA.
Publication TypeJournal Article
Year of Publication2008
AuthorsMinczuk, MA, Papworth, MA, Miller, JC, Murphy, MP, Klug, A
JournalNucleic Acids Res
Volume36
Issue12
Pagination3926-38
Date Published2008 Jul
ISSN1362-4962
KeywordsCell Line, Deoxyribonucleases, Type II Site-Specific, Dimerization, DNA, Mitochondrial, Genetic Vectors, Humans, Mitochondria, Mitochondrial Diseases, Mutation, Peptides, Point Mutation, Protein Engineering, Zinc Fingers
Abstract

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.

DOI10.1093/nar/gkn313
Alternate JournalNucleic Acids Res.
Citation Key10.1093/nar/gkn313
PubMed ID18511461
PubMed Central IDPMC2475635
Grant ListMC_U105184324 / / Medical Research Council / United Kingdom
MC_U105663142 / / Medical Research Council / United Kingdom
/ / Medical Research Council / United Kingdom