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Site-directed recombination via bifunctional PNA-DNA conjugates.
| Content Provider | Semantic Scholar |
|---|---|
| Author | Rogers, Faye A. Vasquez, Karen M. Egholm, Michael Wayland Glazer, Peter M. |
| Copyright Year | 2002 |
| Abstract | 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. |
| File Format | PDF HTM / HTML |
| DOI | 10.1073/pnas.262556899 |
| Alternate Webpage(s) | http://www.pnas.org/content/99/26/16695.full.pdf |
| PubMed reference number | 12461167 |
| Alternate Webpage(s) | https://doi.org/10.1073/pnas.262556899 |
| Volume Number | 99 |
| Issue Number | 26 |
| Journal | Proceedings of the National Academy of Sciences of the United States of America |
| Language | English |
| Access Restriction | Open |
| Content Type | Text |
| Resource Type | Article |
