Search

Mitochondrial DNA (mtDNA) is the only autonomously replicating source of DNA outside the nucleus. The mitochondrial genome encodes thirteen essential polypeptides of the mitochondrial respiratory chain. Defects of the mitochondrial genome can cause severe neurological and multi-systemic disorders. In many patients there is a mixture of mutated and wild-type mtDNA in the same cell (a situation termed heteroplasmy). In these patients the ratio of mutated to wild-type mtDNA is crucial and a biochemical defect only occurs with relatively high levels of mutated mtDNA within an individual cell. This threshold also seems to be critical in the development of mtDNA disease. Since the genetic defect causes a dysfunction in the terminal stage of oxidative metabolism, there is little potential for pharmacological intervention. Molecular techniques must be developed to reverse the ratio of mutated and wild-type mtDNA. In this paper we summarise our approach using both antigenomic peptide nucleic acids and cell necrosis. [ABSTRACT FROM AUTHOR]

Mutations in mitochondrial DNA (mtDNA) cause a spectrum of human pathologies, which predominantly affect skeletal muscle and the central nervous system. In patients, mutated and wild-type mtDNAs often co-exist in the same cell (mtDNA heteroplasmy). In the absence of pharmacological therapy, a genetic strategy for treatment has been proposed whereby replication of mutated mtDNA is inhibited by selective hybridisation of a nucleic acid derivative to the single-stranded replication intermediate, allowing propagation of the wild-type genome and correction of the associated respiratory chain defect. Previous studies have shown the efficacy of this anti-genomic approach in vitro, targeting pathogenic mtDNA templates with only a single point mutation. Pathogenic molecules harbouring deletions, however, present a more difficult problem. Deletions often occur at the site of two short repeat sequences (4-13 residues), only one of which is retained in the deleted molecule. With the more common larger repeats it is therefore difficult to design an anti-genomic molecule that will bind selectively across the breakpoint of the deleted mtDNA. To address this problem, we have used linker-substituted oligodeoxynucleotides to bridge the repeated residues. We show that molecules can be designed to bind more tightly to the deleted as compared to the wild-type mtDNA template, consistent with the nucleotide sequence on either side of the linker co-operating to increase binding affinity. Furthermore, these bridging molecules are capable of sequence-dependent partial inhibition of replication in vitro.

In mammals, mitochondrial DNA (mtDNA) is the only autonomously replicating source of DNA outside the nucleus. Housed in the mitochondrial matrix, this molecule encodes thirteen polypeptides, all of which are believed to be essential components of the mitochondrial respiratory chain. Defects of the mitochondrial genome can cause severe neurological and multi-systemic disorders. As the genetic defect causes a dysfunction in the terminal stage of oxidative metabolism, there is little potential for pharmacological intervention. Thus, there is currently no effective therapy for these chronic progressive disorders. In the disease state, pathogenic mtDNA molecules often cohabit the same cell and tissue with wild type mtDNA, a situation termed heteroplasmy. Manifestation of biochemical and clinical defects occur only when a threshold level of heteroplasmy has been passed. The mitochondrial genome must be continually turned over. Consequently, if a pathogenic mtDNA molecule were to be targeted to prevent it from replicating, the wild type copy would be given a propagative advantage. Over time, therefore, the biochemical and, potentially, the clinical deficiency could be reversed. This manuscript summarises our attempts to identify such an antigenomic molecule, to localise this molecule to mitochondria and to assess its function in whole cells. Finally, we discuss the importance of identifying and designing new antigenomic molecules which may prove effective in treating patients with disorders of the mitochondrial genome.

Although the precise mechanisms of the aging process remain poorly understood, a plausible theory for cellular dysfunction and deterioration during aging involves mitochondria (1, 2). The major function of mitochondria is to generate energy for cellular processes in the form of ATP by oxidative phosphorylation. Mitochondria contain their own DNA (mtDNA), a small 16.5 kb circular molecule that encodes 13 essential polypeptides of the mitochondrial respiratory chain, as well as 2 rRNAs and 22 tRNAs required for intramitochondrial protein synthesis (3). The mitochondrial respiratory chain is a series of five, multisubunit protein complexes located within the inner mitochondrial membrane. The first four of these (complexes I-IV) reoxidize reduced cofactors (NADH and FADH(2)) generated by the oxidation of foodstuffs, thereby generating an electrochemical gradient across the inner mitochondrial membrane which is harnessed by the fifth complex, the ATP synthetase, to drive the formation of ATP.