title: Linked oligodeoxynucleotides show binding cooperativity and can selectively impair replication of deleted mitochondrial DNA templates.
abstract: 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.
authors.affiliation: Department of Neurology, The Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, UK.
authors.forename: R W
authors.initials: RW
authors.lastname: Taylor
authors.forename: T M
authors.initials: TM
authors.lastname: Wardell
authors.forename: B A
authors.initials: BA
authors.lastname: Connolly
authors.forename: D M
authors.initials: DM
authors.lastname: Turnbull
authors.forename: R N
authors.initials: RN
authors.lastname: Lightowlers
mesh_terms: D020029:Base Pairing; D001483:Base Sequence; D019921:Biotinylation; D004261:DNA Replication; D004272:DNA, Mitochondrial / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016678:Genome; D006801:Humans; D007700:Kinetics; D017240:Mitochondrial Myopathies / Q000235:genetics / Q000628:therapy; D009691:Nucleic Acid Denaturation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion / Q000235:genetics*; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance; D013698:Templates, Genetic; D013816:Thermodynamics
chemical_list: D004272:DNA, Mitochondrial; D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides