Mitochondria isolated from liver contain the essential factors required for RNA/DNA oligonucleotide-targeted gene repair

Chimeric RNA/DNA oligonucleotides (ONs) have been used successfully for sit e-specific modifications of episomal and chromosomal DNA in eukaryotic cell s. We explored the possibility of applying this technique to mitochondrial DNA, as single-nucleotide defects in this genome are associated with a seri es of human diseases. Therefore, we determined whether mitochondria possess the enzymatic machinery for chimeric ON-mediated DNA alterations. We utili zed an in vitro DNA repair assay and an Escherichia coil readout system wit h mutagenized plasmids carrying point mutations in antibiotic resistance ge nes. RNA/DNA ONs were designed to correct the defects and restore kanamycin and tetracyclin resistance. Using this system, we demonstrated that extrac ts from highly purified rat liver mitochondria possess the essential enzyma tic activity to mediate precise single-nucleotide changes. Interestingly, t he frequency of gene conversion was similar in both mitochondrial and nucle ar extracts, as well as from quiescent and regenerating liver. The results indicate that mitochondria contain the machinery required for repair of gen omic single-point mutations, and suggest that RNA/DNA ONs may provide a nov el approach to the treatment of certain mitochondrial-based diseases. (C) 2 001 Academic Press.