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.