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

Zongyu Chen, Rod Felsheim, Phillip Wong, Lance B Augustin, Betsy T. Kren, Richard Metz, Clifford J Steer

Research output: Contribution to journalArticlepeer-review

35 Scopus citations

Abstract

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 languageEnglish (US)
Pages (from-to)188-194
Number of pages7
JournalBiochemical and Biophysical Research Communications
Volume285
Issue number2
DOIs
StatePublished - 2001

Keywords

  • Allele-specific PCR
  • Cell-free bacterial assay
  • Gene therapy
  • Mismatch repair
  • Mitochondria
  • Nuclear extract
  • Plasmid
  • RNA/DNA oligonucleotide
  • Single-point mutation
  • Site-directed conversion

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