Bridging PNAs can bind preferentially to a deleted mitochondrial DNA template but replication by mitochondrial DNA polymerase gamma in vitro is not impaired.
Mutations in mitochondrial DNA (mtDNA) are an important cause of neurological and other human pathologies. In the vast majority of cases, supportive care only is available. Mutated and wild-type mtDNAs often coexist in the same cell. A strategy for treatment has been proposed whereby replication of mutated mtDNA is inhibited by selective hybridisation of a nucleic acid derivative, allowing propagation of the wild-type genome and correction of the associated respiratory chain defect. Peptide nucleic acid molecules (PNAs) can be designed to selectively target pathogenic mtDNA with single point mutations. Molecules harbouring deletions present a complex problem. Deletions often occur between two short repeat sequences (4-13 residues), one of which is retained in the deleted molecule. With the more common large repeats, it is therefore difficult to design an antigenomic molecule that will bind selectively under physiological conditions. Following limited success with antigenomic oligodeoxynucleotides (ODNs), we have repeated these studies with a series of bridging PNAs. Molecules complementary to the sequence flanking either side of the 13 bp 'common deletion' were synthesised. The PNAs demonstrated markedly greater affinity for the delete than to the wild-type template. In runoff assays using Klenow fragment, these PNAs selectively inhibited replication of the delete template. However, no selective inhibition was observed when a polymerase gamma-containing mitochondrial fraction was used.
McGregor
Alistair
A
School of Neurology, Neurobiology and Psychiatry, The Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK.
Smith
Paul M
PM
Ross
Günther F
GF
Taylor
Robert W
RW
Turnbull
Douglass M
DM
Lightowlers
Robert N
RN
D001483:Base Sequence; D004256:DNA Polymerase I / Q000378:metabolism; D000074002:DNA Polymerase gamma; D004261:DNA Replication; D004272:DNA, Mitochondrial; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D020135:Peptide Nucleic Acids / Q000378:metabolism*; D017384:Sequence Deletion; D013698:Templates, Genetic
D004272:DNA, Mitochondrial; D020135:Peptide Nucleic Acids; D004256:DNA Polymerase I; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase