Site-specific targeting of psoralen photoadducts with a triple helix-forming oligonucleotide: characterization of psoralen monoadduct and crosslink formation.

A polypurine tract in the supF gene of bacteriophage lambda (base pairs 167-176) was selected as the target for triple helix formation and targeted mutagenesis by an oligopurine (5'-AGGAAGGGGG-3') containing a chemically linked psoralen derivative (4'-hydroxymethyl-4,5',8-trimethylpsoralen) at its 5' terminus (psoAG10). The thymines at base pairs 166 and 167, a 5'ApT site, were targeted for photomodification. Exposure of the triple helical complex to long wavelength ultraviolet radiation led to the covalent binding of psoAG10 to the targeted region in the supF gene and to the induction of site-specific mutations. We report here experiments to characterize the photomodification of the targeted region of the supF gene in the context of triple helix formation. An electrophoretic mobility-shift assay showed that, at low radiation doses, monoadducts at base pair 166 were the major photoadducts. At higher doses the monoadducts were converted to crosslinks between base pairs 166 and 167. HPLC analysis of enzymatically hydrolyzed photoreaction mixtures was used to confirm the electrophoresis results. A strong strand preference for specific photoadduct formation was also detected.     

Triple-helix formation induces recombination in mammalian cells via a nucleotide excision repair-dependent pathway

The ability to stimulate recombination in a site-specific manner in mammalian cells may provide a useful tool for gene knockout and a valuable strategy for gene therapy. We previously demonstrated that psoralen adducts targeted by triple-helix-forming oligonucleotides (TFOs) could induce recombination between tandem repeats of a supF reporter gene in a simian virus 40 vector in monkey COS cells. Based on work showing that triple helices, even in the absence of associated psoralen adducts, are able to provoke DNA repair and cause mutations, we asked whether intermolecular triplexes could stimulate recombination. Here, we report that triple-helix formation itself is capable of promoting recombination and that this effect is dependent on a functional nucleotide excision repair (NER) pathway. Transfection of COS cells carrying the dual supF vector with a purine-rich TFO, AG30, designed to bind as a third strand to a region between the two mutant supF genes yielded recombinants at a frequency of 0.37%, fivefold above background, whereas a scrambled sequence control oligomer was ineffective. In human cells deficient in the NER factor XPA, the ability of AG30 to induce recombination was eliminated, but it was restored in a corrected subline expressing the XPA cDNA. In comparison, the ability of triplex-directed psoralen cross-links to induce recombination was only partially reduced in XPA-deficient cells, suggesting that NER is not the only pathway that can metabolize targeted psoralen photoadducts into recombinagenic intermediates. Interestingly, the triplex-induced recombination was unaffected in cells deficient in DNA mismatch repair, challenging our previous model of a heteroduplex intermediate and supporting a model based on end joining. This work demonstrates that oligonucleotide-mediated triplex formation can be recombinagenic, providing the basis for a potential strategy to direct genome modification by using high-affinity DNA binding ligands.     

Site-directed gene mutation at mixed sequence targets by psoralen-conjugated pseudo-complementary peptide nucleic acids

Sequence-specific DNA-binding molecules such as triple helix-forming oligonucleotides (TFOs) provide a means for inducing site-specific mutagenesis and recombination at chromosomal sites in mammalian cells. However, the utility of TFOs is limited by the requirement for homopurine stretches in the target duplex DNA. Here, we report the use of pseudo-complementary peptide nucleic acids (pcPNAs) for intracellular gene targeting at mixed sequence sites. Due to steric hindrance, pcPNAs are unable to form pcPNA–pcPNA duplexes but can bind to complementary DNA sequences by Watson–Crick pairing via double duplex-invasion complex formation. We show that psoralen-conjugated pcPNAs can deliver site-specific photoadducts and mediate targeted gene modification within both episomal and chromosomal DNA in mammalian cells without detectable off-target effects. Most of the induced psoralen-pcPNA mutations were single-base substitutions and deletions at the predicted pcPNA-binding sites. The pcPNA-directed mutagenesis was found to be dependent on PNA concentration and UVA dose and required matched pairs of pcPNAs. Neither of the individual pcPNAs alone had any effect nor did complementary PNA pairs of the same sequence. These results identify pcPNAs as new tools for site-specific gene modification in mammalian cells without purine sequence restriction, thereby providing a general strategy for designing gene targeting molecules.     

In vivo persistence of DNA triple helices containing psoralen-conjugated oligodeoxyribonucleotides.

Triple helices represent an attractive method for modulating specific gene expression. In particular, cross-linking between a triplex-forming oligonucleotide (TFO) and its duplex DNA target, typically through the formation of psoralen photoadducts, allows efficient blocking of elongation by RNA polymerases in vitro. However, in vivo, this approach is limited by DNA repair of the photoadduct. Here we describe the use of an oligodeoxyribonucleotide 19mer psoralen-modified TFO to form covalent linkages between an oligonucleotide and both strands of the targeted duplex DNA, thereby efficiently blocking expression of a luciferase reporter gene. Most importantly, we demonstrate that both the psoralen cross-link and the purine-motif triplex remained intact for at least 72 h post-transfection, indicating that such species can persist for an extended period of time in vivo. These findings support the feasibility of an antigene approach for the therapeutic regulation of specific gene expression.     

DNA polymerase eta reduces the gamma-H2AX response to psoralen interstrand crosslinks in human cells

DNA interstrand crosslinks are processed by multiple mechanisms whose relationships to each other are unclear. Xeroderma pigmentosum-variant (XP-V) cells lacking DNA polymerase eta are sensitive to psoralen photoadducts created under conditions favoring crosslink formation, suggesting a role for translesion synthesis in crosslink repair. Because crosslinks can lead to double-strand breaks, we monitored phosphorylated H2AX (gamma-H2AX), which is typically generated near double-strand breaks but also in response to single-stranded DNA, following psoralen photoadduct formation in XP-V fibroblasts to assess whether polymerase eta is involved in processing crosslinks. In contrast to conditions favoring monoadducts, conditions favoring psoralen crosslinks induced gamma-H2AX levels in both XP-V and nucleotide excision repair-deficient XP-A cells relative to control repair-proficient cells; ectopic expression of polymerase eta in XP-V cells normalized the gamma-H2AX response. In response to psoralen crosslinking, gamma-H2AX as well as 53BP1 formed coincident foci that were more numerous and intense in XP-V and XP-A cells than in controls. Psoralen photoadducts induced gamma-H2AX throughout the cell cycle in XP-V cells. These results indicate that polymerase eta is important in responding to psoralen crosslinks, and are consistent with a model in which nucleotide excision repair and polymerase eta are involved in processing crosslinks and avoiding gamma-H2AX associated with double-strand breaks and single-stranded DNA in human cells.     

Repair of triple helix directed psoralen adducts in human cells.

Triple helix forming oligonucleotides can direct DNA damaging agents at specific sites in an intact double helix. In our study, triple helix formation was demonstrated in a SV40 based shuttle vector treated with psoralen linked to a 22-mer purine rich oligonucleotide. UVA irradiation caused a covalent linkage of the oligonucleotide through the psoralen to the mutational supF marker gene of the plasmid. After passage in the Jurkat human cell line the recovered vector was analysed in an indicator bacterial strain and mutants were collected. The presence of adducts in the target sequence did not reduce the yield of replicated progeny vector molecules, indicating repair of triple helix associated monoadducts and cross-links. Mutations were highly targeted to a six nucleotide long region of the target sequence. The number of target sequence mutants obtained after triple helix directed psoralen treatment was approximately 160 times higher than with free psoralen. A further investigation of the exact mechanism of the mutational process could make triple helix directed mutagenesis a more useful tool in gene therapy, antiviral therapy, and in studies on DNA repair and genome organisation.     

Potassium-resistant triple helix formation and improved intracellular gene targeting by oligodeoxyribonucleotides containing 7-deazaxanthine.

Triple helix formation by purine-rich oligonucleotides in the anti-parallel motif is inhibited by physiological concentrations of potassium. Substitution with 7-deazaxanthine (c7X) has been suggested as a strategy to overcome this effect. We have tested this by examining triple helix formation both in vitro and in vivo by a series of triple helix-forming oligonucleotides (TFOs) containing guanine plus either adenine, thymine, or c7X. The TFOs were conjugated to psoralen at the 5'end and were designed to bind to a portion of the supF mutation reporter gene. Using in vitro gel mobility shift assays, we found that triplex formation by the c7X-substituted TFOs was relatively resistant to the presence of 140 mM K+. The c7X-containing TFOs were also superior in gene targeting experiments in mammalian cells, yielding 4- to 5-fold higher mutation frequencies in a shuttle vector-based mutagenesis assay designed to detect mutations induced by third strand-directed psoralen adducts. When the phosphodiester backbone was replaced by a phosphorothioate one, the in vitro binding of the c7X-TFOs was not affected, but the efficiency of in vivo triple helix formation was reduced. These results indicate the utility of the c7X substitution for in vivo gene targeting experiments, and they show that the feasibility of the triplex anti-gene strategy can be significantly enhanced by advances in nucleotide chemistry.     

Description of a new amplifiable shuttle vector for mutagenesis studies in human cells: application to N-methyl-N'-nitro-N-nitrosoguanidine-induced mutation spectrum.

In order to analyze the mechanisms of mutagenesis in human cells, we have established a human 293 cell-derived line containing a permanent mutagenesis target, the bacterial lacZ' gene, on an episomal EBV/SV40-based shuttle vector. This plasmid was maintained at a low copy number per cell which rendered it closer to an endogenous gene as compared to the usual transient shuttle vectors. Transient amplification of vectors, inside the host cell due to expression of the SV40 T-antigen, allowed the recovery of a large number of bacterial colonies transformed by plasmids extracted from human cells. Mutations produced in human host cells on the lacZ' locus were easily and rapidly scored and identified in bacteria using the blue/white color assay. Over a 6-month period in culture, we have shown that the lacZ' gene exhibited a low background frequency of point mutations (< 4.8 x 10(-6)). The efficiency of our system for detecting genotoxic-induced mutations was investigated by treating cells with a potent mutagen, the direct alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). A significant increase (< 230-fold) in the frequency of single-base substitutions was observed after MNNG treatment. In total, 63 MNNG-induced independent mutations were characterized. All substitutions but one involved G:C base pairs with 89% being G:C to A:T transitions which is consistent with the MNNG mutagenic specificity already reported in bacteria and mammalian cells. Mutations were distributed along the two strands of the lacZ' gene and there was no obvious influence of either the 5' or the 3' flanking base near the G:C to A:T transition sites. The low spontaneous point mutation frequency on the mutagenesis locus and the ability to detect induced point mutations indicate that this system could be readily used in human mutagenesis studies at the molecular level.     

Repairing the Sickle Cell mutation. II. Effect of psoralen linker length on specificity of formation and yield of third strand-directed photoproducts with the mutant target sequence

Three identical deoxyoligonucleotide third strands with a 3′-terminal psoralen moiety attached by linkers that differ in length (N = 16, 6 and 4 atoms) and structure were examined for their ability to form triplex-directed psoralen photoproducts with both the mutant T residue of the Sickle Cell β-globin gene and the comparable wild-type sequence in linear duplex targets. Specificity and yield of UVA (365 nm) and visible (419 nm) light-induced photoadducts were studied. The total photoproduct yield varies with the linker and includes both monoadducts and crosslinks at various available pyrimidine sites. The specificity of photoadduct formation at the desired mutant T residue site was greatly improved by shortening the psoralen linker. In particular, using the N-4 linker, psoralen interaction with the residues of the non-coding duplex strand was essentially eliminated, while modification of the Sickle Cell mutant T residue was maximized. At the same time, the proportion of crosslink formation at the mutant T residue upon UV irradiation was much greater for the N-4 linker. The photoproducts formed with the wild-type target were fully consistent with its single base pair difference. The third strand with the N-4 linker was also shown to bind to a supercoiled plasmid containing the Sickle Cell mutation site, giving photoproduct yields comparable with those observed in the linear mutant target.     

Directed integration of minute virus of mice DNA into episomes.

Recent studies with adeno-associated virus (AAV) have shown that site-specific integration is directed by DNA sequence motifs that are present in both the viral replication origin and the chromosomal preintegration DNA and that specify binding and nicking sites for the viral regulatory Rep protein. This finding raised the question as to whether other parvovirus regulatory proteins might direct site-specific recombination with DNA targets that contain origin sequences functionally equivalent to those described for AAV. To investigate this question, active and inactive forms of the minute virus of mice (MVM) 3' replication origin, derived from a replicative-form dimer-bridge intermediate, were propagated in an Epstein-Barr virus-based shuttle vector which replicates as an episome in a cell-cycle-dependent manner in mammalian cells. Upon MVM infection of these cells, the infecting genome integrated into episomes containing the active-origin sequence reported to be efficiently nicked by the MVM regulatory protein NS1. In contrast, MVM did not integrate into episomes containing either the inactive form of the origin sequence reported to be inefficiently nicked by NS1 or the active form from which the NS1 consensus nick site had been deleted. The structure of the cloned MVM episomal recombinants displayed several features previously described for AAV episomal and chromosomal recombinants. The findings indicate that the rules which govern AAV site-specific recombination also apply to MVM and suggest that site-specific chromosomal insertions may be achievable with different autonomous parvovirus replicator proteins which recognize binding and nicking sites on the target DNA.     

Shuttle vector plasmid propagation in human peripheral blood lymphocytes facilitated by liposome-mediated transfection

Transfection of human peripheral blood lymphocytes facilitated by a positively charged liposome preparation (Lipofectin, BRL) is 100-fold more efficient than the DEAE dextran technique for the uptake and replication of shuttle vector plasmid DNA. The yield of progeny plasmids obtained from 10 ml of blood was high enough for mutational analysis. A marked increase in the mutation frequency of the shuttle vector marker gene was noted in response to the induction of psoralen adducts in the vector. By using normal human lymphocytes, this method will permit shuttle vector analysis of DNA repair and mutagenesis in a large number of individuals. This method could also prove useful for studies of human lymphotropic viruses.     

Psoralen photo-cross-linking by triplex-forming oligonucleotides at multiple sites in the human rhodopsin gene.

Targeting DNA damage by triplex-forming oligonucleotides (TFOs) represents a way of modifying gene expression and structure and a possible approach to gene therapy. We have determined that this approach can deliver damage with great specificity to sites in the human gene for the G-protein-linked receptor rhodopsin, mutations of which can lead to the genetic disorder autosomal dominant retinitis pigmentosa. We have introduced DNA monoadducts and interstrand cross-links at multiple target sites within the gene using TFOs with a photoactivatable psoralen group at the 5'-end. The extent of formation of photoadducts (i.e., monoadducts and cross-links) was measured at target sites with a 5'-ApT sequence at the triplex-duplex junction and at a target site with 5'-ApT and 5'-TpA sequences located four and seven nucleotides away, respectively. To improve psoralen reactivity at more distant sites, psoralen moieties were attached to TFOs with nucleotide "linkers" from two to nine nucleotides in length. High-affinity binding was maintained with linkers of up to 10 nucleotides, but affinities tended to decrease somewhat with increasing linker length due to faster dissociation kinetics. DNase I footprinting indicated little, if any, interaction between linkers and the duplex. Psoralen-TFO conjugates formed DNA cross-links with high efficiency (56-65%) at 5'-ApT sequences located at triplex junctions. At a 5'-ApT site four nucleotides away, the efficiency varied with linker length; a four-nucleotide linker gave the highest efficiency. Duplexes with 5'-TpA and 5'-ApT sites two nucleotides away, in otherwise identical sequences, were cross-linked with efficiencies of 56 and 38%, respectively. These results indicate that TFO-linker-psoralen conjugates allow simultaneous, efficient targeting of multiple sites in the human rhodopsin gene.     

Targeted mutagenesis in mammalian cells mediated by intracellular triple helix formation.

As an alternative to standard gene transfer techniques for genetic manipulation, we have investigated the use of triple helix-forming oligonucleotides to target mutations to selected genes within mammalian cells. By treating monkey COS cells with oligonucleotides linked to psoralen, we have generated targeted mutations in a simian virus 40 (SV40) vector contained within the cells via intracellular triple helix formation. Oligonucleotide entry into the cells and sequence-specific triplex formation within the SV40 DNA deliver the psoralen to the targeted site. Photoactivation of the psoralen by long-wavelength UV light yields adducts and thereby mutations at that site. We engineered into the SV40 vector novel supF mutation reporter genes containing modified polypurine sites amenable to triplex formation. By comparing the abilities of a series of oligonucleotides to target these new sites, we show that targeted mutagenesis in vivo depends on the strength and specificity of the third-strand binding. Oligonucleotides with weak target site binding affinity or with only partial target site homology were ineffective at inducing mutations in the SV40 vectors within the COS cells. We also show that the targeted mutagenesis is dependent on the oligonucleotide concentration and is influenced by the timing of the oligonucleotide treatment and of the UV irradiation of the cells. Frequencies of intracellular targeted mutagenesis in the range of 1 to 2% were observed, depending upon the conditions of the experiment. DNA sequence analysis revealed that most of the mutations were T.A-to-A.T transversions precisely at the targeted psoralen intercalation site. Several deletions encompassing that site were also seen. The ability to target mutations to selected sites within mammalian cells by using modified triplex-forming oligonucleotides may provide a new research tool and may eventually lead to therapeutic applications.     

Damage distribution and mutation spectrum: the case of 8-methoxypsoralen plus UVA in mammalian cells.

We determined the distribution of monoadducts and biadducts induced in the supF tRNA gene carried by the shuttle vector pZ189, after exposure to 8-methoxypsoralen (8-MOP) plus a double UVA (365 nm) irradiation. These data were compared to our previously published 8-MOP-photoinduced mutation spectrum obtained after propagation of the damaged shuttle vector in mammalian cells. One mutational hot spot in an ATAT/TATA sequence is targeted at a hot spot of biaddition. A second hot spot is not related to the presence of photoadducts either at or near the site. Moreover, it is located in a sequence which can be defined as 'mutation-prone'. Mutations occurring at GC base pairs are not targeted at sites of photoaddition, and may result from a decrease in fidelity of DNA polymerase when copying the damaged vector.     

DNA sequence motifs which direct adeno-associated virus site-specific integration in a model system

The DNA sequence motifs which direct adeno-associated virus type 2 site-specific integration are being investigated using a shuttle vector, propagated as a stable episome in cultured cell lines, as the target for integration. Previously, we reported that the minimum episomal targeting elements comprise a 16-bp binding motif (Rep binding site [RBS]) for a viral regulatory protein (Rep) separated by a short DNA spacer from a sequence (terminal resolution site [TRS]) that can serve as a substrate for Rep-mediated nicking activity (R. M. Linden, P. Ward, C. Giraud, E. Winocour, and K. I. Berns, Proc. Natl. Acad. Sci. USA 93:11288-11294, 1996; R. M. Linden, E. Winocour, and K. I. Berns, Proc. Natl. Acad. Sci. USA 93:7966-7972, 1996). We now report that episomal integration depends upon both the sequence and the position of the spacer DNA separating the RBS and TRS motifs. The spacer thus constitutes a third element required for site-specific episomal integration.     

Sequence-specific protection of duplex DNA against restriction and methylation enzymes by pseudocomplementary PNAs

A new generation of PNAs, so-called pseudocomplementary PNAs (pcPNAs), which are able to target the designated sites on duplex DNA with mixed sequence of purines and pyrimidines via double-duplex invasion mode, has recently been introduced. It has been demonstrated that appropriate pairs of decameric pcPNAs block an access of RNA polymerase to the corresponding promoter. Here, we show that this type of PNAs protects selected DNA sites containing all four nucleobases from the action of restriction enzymes and DNA methyltransferases. We have found that pcPNAs as short as octamers form stable and sequence-specific complexes with duplex DNA in a very salt-dependent manner. In accord with a strand-invasion mode of complex formation, the pcPNA binding proceeds much faster with supercoiled than with linear plasmids. The double-duplex invasion complexes selectively shield specific DNA sites from BclI restriction endonuclease and dam methylase. The pcPNA-assisted protection against enzymatic methylation is more efficient when the PNA-binding site embodies the methylase-recognition site rather than overlaps it. We conclude that pcPNAs may provide the robust tools allowing to sequence-specifically manipulate DNA duplexes in a virtually sequence-unrestricted manner.     

Stability of DNA triplexes on shuttle vector plasmids in the replication pool in mammalian cells

Triple helix-forming oligonucleotides may be useful as gene-targeting reagents in vivo, for applications such as gene knockout. One important property of these complexes is their often remarkable stability, as demonstrated in solution and in cells following transfection. Although encouraging, these measurements do not necessarily report triplex stability in cellular compartments that support DNA functions such as replication and mutagenesis. We have devised a shuttle vector plasmid assay that reports the stability of triplexes on DNA that undergoes replication and mutagenesis. The assay is based on plasmids with novel variant supF tRNA genes containing embedded sequences for triplex formation and psoralen cross-linking. Triple helix-forming oligonucleotides were linked to psoralen and used to form triplexes on the plasmids. At various times after introduction into cells, the psoralen was activated by exposure to long wave ultraviolet light (UVA). After time for replication and mutagenesis, progeny plasmids were recovered and the frequency of plasmids with mutations in the supF gene determined. Site-specific mutagenesis by psoralen cross-links was dependent on precise placement of the psoralen by the triple helix-forming oligonucleotide at the time of UVA treatment. The results indicated that both pyrimidine and purine motif triplexes were much less stable on replicated DNA than on DNA in vitro or in total transfected DNA. Incubation of cells with amidoanthraquinone-based triplex stabilizing compounds enhanced the stability of the pyrimidine triplex.