Chimeric RNA/DNA oligonucleotides (ONs) have been used successfully for site-specific modifications of episomal and chromosomal DNA in eukaryotic cells. We explored the possibility of applying this technique to mitochondrial DNA, as single-nucleotide defects in this genome are associated with a series of human diseases. Therefore, we determined whether mitochondria possess the enzymatic machinery for chimeric ON-mediated DNA alterations. We utilized an in vitro DNA repair assay and an Escherichia coli read-out system with mutagenized plasmids carrying point mutations in antibiotic resistance genes. RNA/DNA ONs were designed to correct the defects and restore kanamycin and tetracyclin resistance, Using this system, we demonstrated that extracts from highly purified rat liver mitochondria possess the essential enzymatic activity to mediate precise single-nucleotide changes. Interestingly, the frequency of gene conversion was similar in both mitochondrial and nuclear extracts, as well as from quiescent and regenerating liver. The results indicate that mitochondria contain the machinery required for repair of genomic single-point mutations, and suggest that RNA/DNA ONs may provide a novel approach to the treatment of certain mitochondrial-based diseases.
|Original language||English (US)|
|Number of pages||7|
|Journal||Biochemical and Biophysical Research Communications|
|State||Published - 2001|
Bibliographical noteFunding Information:
We thank Jeffrey R. Galecke, Cecilia M. P. Rodrigues, and Cheryle L. Linehan-Stieers for excellent technical support and Betsy Rosen-berg and Geoffery Jensen for their assistance with Western blot analysis of the MSH2 protein. This work was supported by National Institutes of Health Grants P01 HL65578-01 (to C.J.S.), P01 HD32652-06 (to B.T.K.), and ValiGen, Inc. (to C.J.S.).
- Allele-specific PCR
- Cell-free bacterial assay
- Gene therapy
- Mismatch repair
- Nuclear extract
- RNA/DNA oligonucleotide
- Single-point mutation
- Site-directed conversion