Site-directed recombination via bifunctional PNA-DNA conjugates.
Site-specific DNA binding molecules offer the potential for genetic manipulation of mammalian cells. Peptide nucleic acids (PNAs) are a DNA mimic in which the purine and pyrimidine bases are attached to a polyamide backbone. PNAs bind with high affinity to single-stranded DNA via Watson-Crick base pairing and can form triple helices via Hoogsteen binding to DNAPNA duplexes. Dimeric bis-PNAs capable of both strand invasion and triplex formation can form clamp structures on target DNAs. As a strategy to promote site-directed recombination, a bis-PNA was coupled to a 40-nt donor DNA fragment homologous to an adjacent region in the target gene. The PNA-DNA conjugate was found to mediate site-directed recombination with a plasmid substrate in human cell-free extracts, resulting in correction of a mutation in a reporter gene at a frequency at least 60-fold above background. Induced site-specific recombination was also seen when the bis-PNA and the donor DNA were co-mixed without covalent linkage. In addition, the bis-PNA and the bis-PNA-DNA conjugate were found to induce DNA repair specifically in the target plasmid. Both the PNA-induced recombination and the PNA-induced repair were found to be dependent on the nucleotide excision repair factor, XPA (xeroderma pigmentosum complementation group A protein). These results suggest that the formation of a PNA clamp on duplex DNA creates a helical distortion that strongly provokes DNA repair and thereby sensitizes the target site to recombination. The ability to promote recombination in a site-directed manner using PNA-DNA conjugates may provide a useful strategy to achieve targeted correction of defective genes.
Rogers
Faye A
FA
Departments of Therapeutic Radiology and Genetics, Yale University School of Medicine, New Haven, CT 06520, USA.
Vasquez
Karen M
KM
Egholm
Michael
M
Glazer
Peter M
PM
D004247:DNA / Q000378:metabolism*; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D020135:Peptide Nucleic Acids / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics*; D051760:Xeroderma Pigmentosum Group A Protein
D004268:DNA-Binding Proteins; D020135:Peptide Nucleic Acids; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein; D004247:DNA