Development of a single-chain, quasi-dimeric zinc-finger nuclease for the selective degradation of mutated human mitochondrial DNA.
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.
Minczuk
Michal
M
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. michal.minczuk@mrc-dunn.cam.ac.uk
Papworth
Monika A
MA
Miller
Jeffrey C
JC
Murphy
Michael P
MP
Klug
Aaron
A
D002460:Cell Line; D004272:DNA, Mitochondrial / Q000737:chemistry / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D005822:Genetic Vectors; D006801:Humans; D008928:Mitochondria / Q000201:enzymology; D028361:Mitochondrial Diseases / Q000235:genetics*; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D017354:Point Mutation; D015202:Protein Engineering; D016335:Zinc Fingers
D004272:DNA, Mitochondrial; D010455:Peptides; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific