Site-directed recombination via bifunctional PNA-DNA conjugates

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.

Fig 1.

Design and synthesis of a bifunctional PNA–DNA conjugate. (A) Sequence of and predicted clamp formation by the bis-PNA designed to bind to the homopurine strand at positions 167–176 of the supFG1 gene. (B) Orientation of triple helix formation by the TFO, AG30, which binds to bp 167–196 of the supFG1 gene. (C) Preparation of the PNA–DNA bifunctional molecule. The DNA oligonucleotide-bis-PNA conjugation was carried out by means of reaction between the 3′-thiol-modified oligonucleotide and the maleimido-modified bis-PNA. a, 0.1 M DTT; b, 0.1 M phosphate buffer (pH 7.0). (D) Analysis of PNA–DNA conjugate by denaturing 12% PAGE (7 M urea). Lane 1, DNA donor fragment A; lane 2, A–PNA hybrid molecule after purification by preparative gel electrophoresis.

Fig 2.

Targeted recombination induced by PNA clamp formation. (A) Design of the plasmid vector assay to detect targeted recombination in the supFG1 reporter gene by PNA–DNA conjugates and related molecules. (B) PNA-induced recombination in vitro in HeLa cell-free extracts. The pSupFG1/G144C plasmid DNA was incubated in vitro with the indicated PNAs or oligonucleotides in the presence or absence of human cell-free extracts, as indicated. After 2 h, the plasmid DNA was isolated and used to transform indicator bacteria for genetic analysis of the supFG1 gene. The bars indicate the frequency of blue colonies (representing recombinants) out of total colonies, with the actual count given to the right of each bar. The samples A–AG30 and A–PNA indicate molecules in which the donor DNA fragment, A, was covalently linked either to the TFO, AG30, or to the bis-PNA, respectively. The sample A + PNA represents the donor DNA fragment and the bis-PNA mixed together as separate, unlinked molecules. Error bars indicate standard errors.

Fig 3.

DNA repair synthesis stimulated by the formation of a PNA clamp in HeLa cell-free extracts. Supercoiled plasmids pSupFG1 and pIND/lacZ were preincubated at 37°C with PNA and TFO oligomers to allow binding via either clamp or triplex formation. The DNA was then added to HeLa extracts supplemented with [α-³²P]dCTP. After 3 h at 30°C, the reactions were terminated and the plasmid DNAs were isolated, linearized by EcoRI digestion, and analyzed by agarose gel electrophoresis. (A) Visualization of the plasmid DNA by ethidium bromide staining. (B) Autoradiogram showing labeled nucleotide incorporation indicative of DNA repair synthesis. (C) Quantification of incorporation of [α-³²P]dCTP into plasmid DNA. Each bar represents the ratio of intensity of the pSupFG1 band over the intensity of the control pIND/lacZ band representing background. The absolute incorporation was then normalized by setting the value for the UV-induced damage at 100%. The data are the average obtained from three individual experiments, with error bars calculated as the standard error.

Fig 4.

Role of the NER pathway in PNA clamp-induced repair synthesis. DNA repair synthesis in HeLa cell-free extracts was measured as in Fig. 3. pSupFG1 plasmid DNA was untreated (control), treated with UV light, or incubated with the bis-PNA, as indicated. Extract samples were unmodified, XPA-depleted (−XPA), or XPA-depleted followed by supplementation with recombinant XPA protein (+XPA). (A) Immunodepletion of the NER factor, XPA, from HeLa cell-free extracts. Extract samples were immunodepleted using a rabbit polyclonal XPA antibody premixed with protein A agarose beads (25, 28). The immunoprecipitate was removed by centrifugation, and the remaining supernatant was examined by Western blot analysis. (B) Visualization by ethidium bromide staining of plasmid DNAs isolated from the extract reactions. (C) Autoradiogram showing labeled nucleotide incorporation indicative of repair synthesis. (D) Quantification of incorporation of [α-³²P]dCTP into plasmid DNA. The amount of incorporation in each case was normalized by setting the value for the UV-induced damage at 100%. The data are the average obtained from three individual experiments, with error bars calculated as the standard error.

Fig 5.

Role of the NER pathway in PNA clamp-induced recombination. Targeted recombination induced by PNA clamp formation was assayed in vitro in HeLa cell-free extracts as in Fig. 2. The pSupFG1/G144C plasmid DNA was incubated with the indicated PNAs and oligonucleotides in extract samples that were complete, XPA-depleted (−XPA), or XPA-depleted followed by supplementation with recombinant XPA protein (+XPA). After 2 h, the plasmid DNA was isolated and used to transform indicator bacteria for genetic analysis of the supFG1 gene. The bars indicate the frequency of blue colonies (representing recombinants) out of total colonies, with the actual count given to the right of each bar. A–PNA indicates molecules in which the donor DNA fragment, A, was covalently linked either to the bis-PNA, whereas the sample A + PNA represents the donor DNA fragment and the bis-PNA mixed together as separate, unlinked molecules. Error bars indicate standard errors.