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 Posted: Fri Oct 03, 2008 9:22 pm Post subject: A SENS strategy fulfilled. Development of a single-chain, q |
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Nucleic Acids Res. 2008 Jul;36(12):3926-38. Epub 2008 May 29. Links
Development of a single-chain, quasi-dimeric zinc-finger nuclease for
the selective degradation of mutated human mitochondrial DNA.Minczuk
M, Papworth MA, Miller JC, Murphy MP, Klug A.
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH,
UK. michal.minczuk@mrc-dunn.cam.ac.uk
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.
PMID: 18511461 [PubMed - indexed for MEDLINE]
PMCID: PMC2475635 |
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