RESUMEN
Gene amplification drives oncogenesis in a broad spectrum of cancers. A number of drugs have been developed to inhibit the protein products of amplified driver genes, but their clinical efficacy is often hampered by drug resistance. Here, we introduce a therapeutic strategy for targeting cancer-associated gene amplifications by activating the DNA damage response with triplex-forming oligonucleotides (TFOs), which drive the induction of apoptosis in tumors, whereas cells without amplifications process lower levels of DNA damage. Focusing on cancers driven by HER2 amplification, we find that TFOs targeting HER2 induce copy number-dependent DNA double-strand breaks (DSBs) and activate p53-independent apoptosis in HER2-positive cancer cells and human tumor xenografts via a mechanism that is independent of HER2 cellular function. This strategy has demonstrated in vivo efficacy comparable to that of current precision medicines and provided a feasible alternative to combat drug resistance in HER2-positive breast and ovarian cancer models. These findings offer a general strategy for targeting tumors with amplified genomic loci.
Asunto(s)
Neoplasias de la Mama , Amplificación de Genes , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Daño del ADN , Femenino , Genómica , Humanos , OligonucleótidosRESUMEN
Exploitation of DNA repair defects has enabled major advances in treating specific cancers. Recent work discovered that the oncometabolite 2-hydroxyglutarate (2-HG), produced by neomorphic isocitrate dehydrogenase 1/2 (IDH1/2) mutations, confers a homology-directed repair (HDR) defect through 2-HG-induced histone hypermethylation masking HDR signaling. Here, we report that IDH1-mutant cancer cells are profoundly sensitive to the histone deacetylase inhibitor (HDACi) vorinostat, by further suppressing the residual HDR in 2-HG-producing cells. Vorinostat downregulates repair factors BRCA1 and RAD51 via disrupted E2F-factor regulation, causing increased DNA double-strand breaks, reduced DNA repair factor foci, and functional HDR deficiency even beyond 2-HG's effects. This results in greater cell death of IDH1-mutant cells and confers synergy with radiation and PARPi, both against cells in culture and patient-derived tumor xenografts. Our work identifies HDACi's utility against IDH1-mutant cancers, and presents IDH1/2 mutations as potential biomarkers to guide trials testing HDACi in gliomas and other malignancies. IMPLICATIONS: IDH1-mutant cells show profound vulnerability to HDACi treatment, alone and with PARPi and radiation, via HDR suppression, presenting IDH1/2 mutations as biomarkers for HDACi use in gliomas and other malignancies.
Asunto(s)
Reparación del ADN/genética , Glioma/tratamiento farmacológico , Inhibidores de Histona Desacetilasas/uso terapéutico , Isocitrato Deshidrogenasa/metabolismo , Animales , Línea Celular Tumoral , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Ratones , Ratones DesnudosRESUMEN
Disruption of the tumor suppressor PTEN, either at the protein or genomic level, plays an important role in human cancer development. The high frequency of PTEN deficiency reported across several cancer subtypes positions therapeutic approaches that exploit PTEN loss-of-function with the ability to significantly impact the treatment strategies of a large patient population. Here, we report that an endophytic fungus isolated from a medicinal plant produces an inhibitor of DNA double-strand-break repair. Furthermore, the novel alkaloid product, which we have named irrepairzepine (1), demonstrated synthetic lethal targeting in PTEN-deficient glioblastoma cells. Our results uncover a new therapeutic lead for PTEN-deficient cancers and an important molecular tool toward enhancing the efficacy of current cancer treatments.
Asunto(s)
Neoplasias Encefálicas/tratamiento farmacológico , Reparación del ADN/efectos de los fármacos , Endófitos/química , Glioblastoma/tratamiento farmacológico , Fosfohidrolasa PTEN/genética , Mutaciones Letales Sintéticas/genética , Neoplasias Encefálicas/genética , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Ensayo Cometa , Roturas del ADN de Doble Cadena/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Ecuador , Glioblastoma/genética , Humanos , Estructura Molecular , Mutágenos/toxicidad , Ensayo de Tumor de Célula MadreRESUMEN
Structural alterations in DNA can serve as natural impediments to replication fork stability and progression, resulting in DNA damage and genomic instability. Naturally occurring polypurine mirror repeat sequences in the human genome can create endogenous triplex structures evoking a robust DNA damage response. Failures to recognize or adequately process these genomic lesions can result in loss of genomic integrity. Nucleotide excision repair (NER) proteins have been found to play a prominent role in the recognition and repair of triplex structures. We demonstrate using triplex-forming oligonucleotides that chromosomal triplexes perturb DNA replication fork progression, eventually resulting in fork collapse and the induction of double strand breaks (DSBs). We find that cells deficient in the NER damage recognition proteins, XPA and XPC, accumulate more DSBs in response to chromosomal triplex formation than NER-proficient cells. Furthermore, we demonstrate that XPC-deficient cells are particularly prone to replication-associated DSBs in the presence of triplexes. In the absence of XPA or XPC, deleterious consequences of triplex-induced genomic instability may be averted by activating apoptosis via dual phosphorylation of the H2AX protein. Our results reveal that damage recognition by XPC and XPA is critical to maintaining replication fork integrity and preventing replication fork collapse in the presence of triplex structures.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , Replicación del ADN , Fibroblastos/metabolismo , Conformación de Ácido Nucleico , Animales , Apoptosis , Línea Celular , Proteínas de Unión al ADN/metabolismo , Inestabilidad Genómica , Ratones Transgénicos , Oligonucleótidos/química , Fosforilación , Recombinasa Rad51/metabolismo , Proteína de Replicación A/metabolismo , Proteína de la Xerodermia Pigmentosa del Grupo A/metabolismoRESUMEN
Monogenic skin diseases arise from well-defined single gene mutations, and in some cases a single point mutation. As the target cells are superficial, these diseases are ideally suited for treatment by nucleic acid-based therapies as well as monitoring through a variety of noninvasive imaging technologies. Despite the accessibility of the skin, there remain formidable barriers for functional delivery of nucleic acids to the target cells within the dermis and epidermis. These barriers include the stratum corneum and the layered structure of the skin, as well as more locally, the cellular, endosomal and nuclear membranes. A wide range of technologies for traversing these barriers has been described and moderate success has been reported for several approaches. The lessons learned from these studies include the need for combinations of approaches to facilitate nucleic acid delivery across these skin barriers and then functional delivery across the cellular and nuclear membranes for expression (e.g., reporter genes, DNA oligonucleotides or shRNA) or into the cytoplasm for regulation (e.g., siRNA, miRNA, antisense oligos). The tools for topical delivery that have been evaluated include chemical, physical and electrical methods, and the development and testing of each of these approaches has been greatly enabled by imaging tools. These techniques allow delivery and real time monitoring of reporter genes, therapeutic nucleic acids and also triplex nucleic acids for gene editing. Optical imaging is comprised of a number of modalities based on properties of light-tissue interaction (e.g., scattering, autofluorescence, and reflectance), the interaction of light with specific molecules (e.g., absorbtion, fluorescence), or enzymatic reactions that produce light (bioluminescence). Optical imaging technologies operate over a range of scales from macroscopic to microscopic and if necessary, nanoscopic, and thus can be used to assess nucleic acid delivery to organs, regions, cells and even subcellular structures. Here we describe the animal models, reporter genes, imaging approaches and general strategies for delivery of nucleic acids to cells in the skin for local expression (e.g., plasmid DNA) or gene silencing (e.g., siRNA) with the intent of developing nucleic acid-based therapies to treat diseases of the skin.
Asunto(s)
Técnicas de Transferencia de Gen , Imagen Molecular/métodos , Ácidos Nucleicos/genética , Piel/metabolismo , Animales , Modelos Animales de Enfermedad , Expresión Génica , Genes Reporteros , Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/terapia , Humanos , Mediciones Luminiscentes/métodos , Ratones , Ratones Transgénicos , Microscopía/métodos , Plásmidos/administración & dosificación , Plásmidos/genética , ARN Interferente Pequeño/genética , Enfermedades de la Piel/genética , Enfermedades de la Piel/terapiaRESUMEN
Triplex structures generated by sequence-specific triplex-forming oligonucleotides (TFOs) have proven to be promising tools for gene targeting strategies. In addition, triplex technology has been highly utilized to study the molecular mechanisms of DNA repair, recombination and mutagenesis. However, triplex formation utilizing guanine-rich oligonucleotides as third strands can be inhibited by potassium-induced self-association resulting in G-quadruplex formation. We report here that guanine-rich TFOs partially substituted with 8-aza-7-deaza-guanine (PPG) have improved target site binding in potassium compared with TFOs containing the natural guanine base. We designed PPG-substituted TFOs to bind to a polypurine sequence in the supFG1 reporter gene. The binding efficiency of PPG-substituted TFOs to the target sequence was analyzed using electrophoresis mobility gel shift assays. We have determined that in the presence of potassium, the non-substituted TFO, AG30 did not bind to its target sequence, however binding was observed with the PPG-substituted AG30 under conditions with up to 140 mM KCl. The PPG-TFOs were able to maintain their ability to induce genomic modifications as measured by an assay for gene-targeted mutagenesis. In addition, these compounds were capable of triplex-induced DNA double strand breaks, which resulted in activation of apoptosis.
Asunto(s)
ADN/química , Nucleósidos/química , Oligonucleótidos/química , Pirimidinonas/química , Animales , Sitios de Unión , ADN/genética , ADN/metabolismo , Roturas del ADN de Doble Cadena , Marcación de Gen , Genes Reporteros , Guanina/química , Ratones , Mutagénesis , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Potasio/químicaRESUMEN
Cellular DNA damage response is critical to preserving genomic integrity following exposure to genotoxic stress. A complex series of networks and signaling pathways become activated after DNA damage and trigger the appropriate cellular response, including cell cycle arrest, DNA repair, and apoptosis. The response elicited is dependent upon the type and extent of damage sustained, with the ultimate goal of preventing propagation of the damaged DNA. A major focus of our studies is to determine the cellular pathways involved in processing damage induced by altered helical structures, specifically triplexes. Our lab has demonstrated that the TFIIH factor XPD occupies a central role in triggering apoptosis in response to triplex-induced DNA strand breaks. We have shown that XPD co-localizes with γH2AX, and its presence is required for the phosphorylation of H2AX tyrosine142, which stimulates the signaling pathway to recruit pro-apoptotic factors to the damage site. Herein, we examine the cellular pathways activated in response to triplex formation and discuss our finding that suggests that XPD-dependent apoptosis plays a role in preserving genomic integrity in the presence of excessive structurally induced DNA damage.
Asunto(s)
Apoptosis/genética , Daño del ADN , Reparación del ADN , ADN/genética , Transducción de Señal/genética , Supervivencia Celular/genética , ADN/química , ADN/metabolismo , Células HeLa , Histonas/metabolismo , Humanos , Modelos Genéticos , Fosforilación , Proteína de la Xerodermia Pigmentosa del Grupo D/metabolismoRESUMEN
DNA sequences capable of forming triplexes are prevalent in the human genome and have been found to be intrinsically mutagenic. Consequently, a balance between DNA repair and apoptosis is critical to counteract their effect on genomic integrity. Using triplex-forming oligonucleotides to synthetically create altered helical distortions, we have determined that pro-apoptotic pathways are activated by the formation of triplex structures. Moreover, the TFIIH factor, XPD, occupies a central role in triggering apoptosis in response to triplex-induced DNA strand breaks. Here, we show that triplexes are capable of inducing XPD-independent double strand breaks, which result in the formation of γH2AX foci. XPD was subsequently recruited to the triplex-induced double strand breaks and co-localized with γH2AX at the damage site. Furthermore, phosphorylation of H2AX tyrosine 142 was found to stimulate the signaling pathway of XPD-dependent apoptosis. We suggest that this mechanism may play an active role in minimizing genomic instability induced by naturally occurring noncanonical structures, perhaps protecting against cancer initiation.
Asunto(s)
Apoptosis , Daño del ADN , ADN , Proteína de la Xerodermia Pigmentosa del Grupo D/metabolismo , Animales , Línea Celular , ADN/química , Roturas del ADN de Doble Cadena , Reparación del ADN , Inestabilidad Genómica , Ratones , Proteína de la Xerodermia Pigmentosa del Grupo D/fisiologíaRESUMEN
Epidermal keratinocytes are particularly suitable candidates for in situ gene correction. Intraperitoneal administration of a triplex-forming oligonucleotide (TFO) was previously shown to introduce DNA base changes in a reporter gene in skin, without identifying which cells had been targeted. We extend those previous experiments using two triplex-forming molecules, a peptide nucleic acid-antennapedia (PNA-Antp), and a TFO (AG30), as well as two lines of transgenic mice that have the chromosomally integrated λsupFG1 shuttle-reporter transgene. Successful in vivo genomic modification occurs in the epidermis and dermis in CD1 transgenic mice following either intraperitoneal or intradermal delivery of the PNA-Antp conjugate. FITC-PNA-Antp accumulates in nuclei of keratinocytes, and, after intradermal delivery of the PNA-Antp, chromosomally modified, keratin 5-positive basal keratinocytes persist for at least 10 days. In hairless (SKH1) mice with the λsupFG1 transgene, intradermal delivery of the TFO, AG30, introduces gene modifications in both tail and back skin, and these chromosomal modifications persist in basal keratinocytes for 10 days. Hairless mice should facilitate comparison of various targeting agents and methods of delivery. Gene targeting by repeated local administration of oligonucleotides may prove clinically useful for judiciously selected disease-causing genes in the epidermis.
Asunto(s)
ADN/metabolismo , Epidermis/metabolismo , Marcación de Gen/métodos , Oligonucleótidos/administración & dosificación , Ácidos Nucleicos de Péptidos/administración & dosificación , Animales , Proteína con Homeodominio Antennapedia/administración & dosificación , Proteína con Homeodominio Antennapedia/farmacología , Péptidos Catiónicos Antimicrobianos/administración & dosificación , Péptidos Catiónicos Antimicrobianos/farmacología , ADN/efectos de los fármacos , Inyecciones Intradérmicas , Inyecciones Intraperitoneales , Ratones , Ratones Pelados , Ratones Endogámicos , Ratones Transgénicos , Modelos Animales , Oligonucleótidos/farmacología , Ácidos Nucleicos de Péptidos/farmacologíaRESUMEN
Hematopoietic stem cell (HSC) gene therapy offers promise for the development of new treatments for a variety of hematologic disorders. However, efficient in vivo modification of HSCs has proved challenging, thus imposing constraints on the therapeutic potential of this approach. Herein, we provide a gene-targeting strategy that allows site-specific in vivo gene modification in the HSCs of mice. Through conjugation of a triplex-forming peptide nucleic acid (PNA) to the transport peptide, antennapedia (Antp), we achieved successful in vivo chromosomal genomic modification of hematopoietic progenitor cells, while still retaining intact differentiation capabilities. Following systemic administration of PNA-Antp conjugates, sequence-specific gene modification was observed in multiple somatic tissues as well as within multiple compartments of the hematopoietic system, including erythroid, myeloid, and lymphoid cell lineages. As a true functional measure of gene targeting in a long-term renewable HSC, we also demonstrate preserved genomic modification in the bone marrow and spleen of primary recipient mice following transplantation of bone marrow from PNA-Antp-treated donor mice. Our approach offers a minimally invasive alternative to ex vivo gene therapy, by eliminating the need for the complex steps of stem cell mobilization and harvesting, ex vivo manipulation, and transplantation of stem cells. Therefore, our approach may provide new options for individualized therapies in the treatment of monogenic hematologic diseases such as sickle cell anemia and thalassemia.
Asunto(s)
Células Madre Hematopoyéticas/metabolismo , Ácidos Nucleicos de Péptidos/administración & dosificación , Animales , Células de la Médula Ósea/metabolismo , Diferenciación Celular , Línea Celular , Linaje de la Célula/genética , Femenino , Marcación de Gen , Técnicas de Transferencia de Gen , Terapia Genética , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Inyecciones Intraperitoneales , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Ácidos Nucleicos de Péptidos/química , Ácidos Nucleicos de Péptidos/metabolismoRESUMEN
Peptide nucleic acids (PNAs) bind duplex DNA in a sequence-specific manner, creating triplex structures that can provoke DNA repair and produce genome modification. CCR5 encodes a chemokine receptor required for HIV-1 entry into human cells, and individuals carrying mutations in this gene are resistant to HIV-1 infection. Transfection of human cells with PNAs targeted to the CCR5 gene, plus donor DNAs designed to introduce stop codons mimicking the naturally occurring CCR5-delta32 mutation, produced 2.46% targeted gene modification. CCR5 modification was confirmed at the DNA, RNA, and protein levels and was shown to confer resistance to infection with HIV-1. Targeting of CCR5 was achieved in human CD34(+) hematopoietic stem cells (HSCs) with subsequent engraftment into mice and persistence of the gene modification more than four months posttransplantation. This work suggests a therapeutic strategy for CCR5 knockout in HSCs from HIV-1-infected individuals, rendering cells resistant to HIV-1 and preserving immune system function.
Asunto(s)
Células Madre Hematopoyéticas/metabolismo , Ácidos Nucleicos de Péptidos/farmacología , Receptores CCR5/metabolismo , Animales , Antígenos CD34/metabolismo , Secuencia de Bases , Sitios de Unión , Antagonistas de los Receptores CCR5 , Línea Celular , Codón de Terminación , Reparación del ADN , Marcación de Gen/métodos , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , VIH-1/fisiología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/virología , Humanos , Ratones , Mutación , Ácidos Nucleicos de Péptidos/química , Receptores CCR5/genéticaRESUMEN
Beta-thalassemia is a genetic disorder caused by mutations in the beta-globin gene. Triplex-forming oligonucleotides and triplex-forming peptide nucleic acids (PNAs) have been shown to stimulate recombination in mammalian cells via site-specific binding and creation of altered helical structures that provoke DNA repair. However, the use of these molecules for gene targeting requires homopurine tracts to facilitate triple helix formation. Alternatively, to achieve binding to mixed-sequence target sites for the induced gene correction, we have used pseudo-complementary PNAs (pcPNAs). Due to steric hindrance, pcPNAs are unable to form pcPNA-pcPNA duplexes but can bind to complementary DNA sequences via double duplex-invasion complexes. We demonstrate here that pcPNAs, when co-transfected with donor DNA fragments, can promote single base pair modification at the start of the second intron of the beta-globin gene. This was detected by the restoration of proper splicing of transcripts produced from a green fluorescent protein-beta globin fusion gene. We also demonstrate that pcPNAs are effective in stimulating recombination in human fibroblast cells in a manner dependent on the nucleotide excision repair factor, XPA. These results suggest that pcPNAs can be effective tools to induce heritable, site-specific modification of disease-related genes in human cells without purine sequence restriction.
Asunto(s)
Marcación de Gen/métodos , Mutación , Ácidos Nucleicos de Péptidos/química , Talasemia/genética , Globinas beta/genética , Animales , Células CHO , Línea Celular , Cricetinae , Cricetulus , Humanos , Plásmidos/genética , Recombinación Genética , Fase S , Proteína de la Xerodermia Pigmentosa del Grupo A/metabolismoRESUMEN
Triplex-forming oligonucleotides (TFOs) bind in the major groove of duplex DNA at polypurine/ polypyrimidine stretches in a sequence-specific manner. The binding specificity of TFOs makes them potential candidates for use in directed genome modification. A number of studies have shown that TFOs can introduce permanent changes in a target sequence by stimulating a cell's inherent repair pathways. TFOs have also been demonstrated to inhibit gene expression providing a possible role for these compounds in cancer therapy. This review summarizes the dual roles of TFOs for use in delivering DNA reactive compounds to a specific site in the genome or for introducing permanent changes in the target sequence through the introduction of an altered helical structure. In addition to compiling the ways in which TFOs have been successfully utilized, this review will explore conflicting reports of TFO bioactivity focusing on the variables which affect the efficacy in vitro of TFO mediated genomic modification which in turn may represent the obstacles encountered using TFOs to modulate gene expression in vivo.
Asunto(s)
ADN/química , ADN/genética , Regulación de la Expresión Génica/genética , Conformación de Ácido Nucleico , Oligonucleótidos/química , Oligonucleótidos/genética , Animales , Humanos , Mutágenos/química , Mutágenos/farmacologíaRESUMEN
Triplex-forming oligonucleotides (TFOs) have the potential to serve as gene therapeutic agents on the basis of their ability to mediate site-specific genome modification via induced recombination. However, high-affinity triplex formation is limited to polypurine/polypyrimidine sites in duplex DNA. Because of this sequence restriction, careful analysis is needed to identify suitable TFO target sites within or near genes of interest. We report here an examination of two key parameters which influence the efficiency of TFO-induced recombination: (1) binding affinity of the TFO for the target site and (2) the distance between the target site and the mutation to be corrected. To test the influence of binding affinity, we compared induced recombination in human cell-free extracts by a series of G-rich oligonucleotides with an identical base composition and an increasing number of mismatches in the third strand binding code. As the number of mismatches increased and, therefore, binding affinity decreased, induced recombination frequency also dropped. There was an apparent threshold at an equilibrium dissociation constant (K(d)) of 1 x 10(-)(7) M. In addition, TFO chemical modification with N,N-diethylethylenediamine (DEED) internucleoside linkages to confer improved binding was found to yield increased levels of induced recombination. To test the ability of triplex formation to induce recombination at a distance, episomal targets with informative reporter genes were constructed to contain polypurine TFO target sites at varying distances from the mutations to be corrected. TFO-induced recombination in mammalian cells between a plasmid vector and a donor oligonucleotide was detected at distances ranging from 24 to 750 bp. Together, these results indicate that TFO-induced recombination requires high-affinity binding but can affect sites hundreds of base pairs away from the position of triplex formation.
Asunto(s)
ADN/genética , ADN/metabolismo , Conformación de Ácido Nucleico , Recombinación Genética , Animales , Disparidad de Par Base/genética , Sitios de Unión , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , ADN/síntesis química , Reparación del ADN/genética , Ensayo de Cambio de Movilidad Electroforética , Etilenodiaminas/química , Vectores Genéticos , Guanina/metabolismo , Células HeLa , Humanos , Ácidos Nucleicos Heterodúplex/síntesis química , Ácidos Nucleicos Heterodúplex/genética , Ácidos Nucleicos Heterodúplex/metabolismo , Oligonucleótidos/síntesis química , Oligonucleótidos/metabolismo , TransfecciónRESUMEN
Triplex-forming oligonucleotides (TFOs) are DNA-binding molecules, which offer the potential to selectively modulate gene expression. However, the biological activity of TFOs as potential antigene compounds has been limited by cellular uptake. Here, we investigate the effect of cell-penetrating peptides on the biological activity of TFOs as measured in an assay for gene-targeted mutagenesis. Using the transport peptide derived from the third helix of the homeodomain of antennapedia (Antp), we tested TFO-peptide conjugates compared with unmodified TFOs. TFOs covalently linked to Antp resulted in a 20-fold increase in mutation frequency when compared with 'naked' oligonucleotides. There was no increase above background in mutation frequency when Antp by itself was added to the cells or when Antp was linked to mixed or scrambled sequence control oligonucleotides. In addition, the TFO-peptide conjugates increased the mutation frequency of the target gene, and not the control gene, in a dose-responsive manner. Confocal microscopy using labeled oligonucleotides indicated increased cellular uptake of TFOs when linked to Antp, consistent with the gene-targeting data. These results suggest that peptide conjugation may enhance intranuclear delivery of reagents designed to bind to chromosomal DNA.
Asunto(s)
Cromosomas/efectos de los fármacos , Marcación de Gen , Proteínas de Homeodominio/química , Proteínas Nucleares/química , Oligonucleótidos/química , Oligonucleótidos/farmacología , Factores de Transcripción/química , Animales , Proteína con Homeodominio Antennapedia , Transporte Biológico , Línea Celular , ADN/química , Ratones , Microscopía Confocal , Mutagénesis , Mutación , Oligonucleótidos/metabolismoRESUMEN
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.