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Triplex-forming oligonucleotides (TFOs) recognize and bind to specific duplex DNA sequences and have been used extensively to modify gene function in cells. Although germ line mutations can be incorporated by means of embryonic stem cell technology, little progress has been made toward introducing mutations in somatic cells of living organisms. Here we demonstrate that TFOs can induce mutations at specific genomic sites in somatic cells of adult mice. Mutation detection was facilitated by the use of transgenic mice bearing chromosomal copies of the supF and cII reporter genes. Mice treated with a supF-targeted TFO displayed about fivefold greater mutation frequencies in the supF gene compared with mice treated with a scrambled sequence control oligomer. No mutagenesis was detected in the control gene (cII) with either oligonucleotide. These results demonstrate that site-specific, TFO-directed genome modification can be accomplished in intact animals.

Specific recognition of a region of duplex DNA by triplex-forming oligonucleotides (TFOs) provides an attractive strategy for genetic manipulation. Based on this, we have investigated the ability of the triplex-directed approach to induce mutations at a chromosomal locus in living cells. A mouse fibroblast cell line was constructed containing multiple chromosomal copies of the lambdasupFG1 vector carrying the supFG1 mutation-reporter gene. Cells were treated with specific (psoAG30) or control (psoSCR30) psoralen-conjugated TFOs in the presence and absence of UVA irradiation. The results demonstrated a 6- to 10-fold induction of supFG1 mutations in the psoAG30-treated cells as compared with psoSCR30-treated or untreated control cells. Interestingly, UVA irradiation had no effect onthe mutation frequencies induced by the psoralen-conjugated TFOs, suggesting a triplex-mediated but photoproduct-independent process of mutagenesis. Sequencing data were consistent with this finding since the expected T.A-->A.T transversions at the predicted psoralen crosslinking site were not detected. However, insertions and deletions were detected within the triplex binding site, indicating a TFO-specific induction of mutagenesis. This result demonstrates the ability of triplex-forming oligonucleotides to influence mutation frequencies at a specific site in a mammalian chromosome.

We have explored the application of triplex technology to the human rhodopsin gene, which encodes a G-protein-linked receptor involved in the genetic disorder autosomal dominant retinitis pigmentosa (ADRP). Our results support the hypothesis that most human genes contain high-affinity triplex sites and further refine the rules governing identification and successful targeting of triplex-forming oligonucleotides (TFOs) to these sites. Using a computer search for sites 15 nucleotides in length and greater than 80% purine, we found 143 distinct sites in the rhodopsin gene and comparable numbers of sites in several other human genes. By applying more stringent criteria, we selected 17 potential target sites in the rhodopsin gene, screened them with a plasmid binding assay, and found 8 that bound TFOs with submicromolar affinity (Kd = 10(-)9-10(-)7 M). We compared purine (GA) and mixed (GT) TFOs at each site, and found that GA-TFOs consistently bound with higher affinity, and were less sensitive to pyrimidine interruptions in the target strand. High G-content favored high-affinity binding; only sites with >54% G-content bound TFOs with Kd

Targeting damage to specific sites in the genome represents an attractive approach to manipulating gene function in mammalian cells. To test the applicability of triple-helix formation as a means for achieving precisely timed site-specific damage within a mammalian gene, a triplex-forming oligodeoxyribonucleotide (TFO) that binds with high affinity to a specific site within the hamster adenine phosphoribosyltransferase (APRT) gene was modified with the photochemically reactive psoralen derivative 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT). The modified TFO, psorTFO1, bound with high affinity to a target site within intron 1 of the APRT gene. Upon irradiation, photomonoadducts (i.e., covalent adducts of psorTFO1 to one strand of the target duplex) were formed with high efficiency (approximately 50%). Introduction of 5'-TpA sequences (the preferred site for psoralen-induced photo-cross-links) at or near the triplex junction leads to increased efficiency of total photoadduct formation and to efficient formation of products that had the electrophoretic mobility on denaturing PAGE expected for three-stranded photo-cross-links (i.e., products containing psorTFO1 covalently linked to both strands of the duplex). Their identities as cross-links were verified by (1) identical electrophoretic mobility of products formed with either duplex strand radiolabeled and (2) coprecipitation of the radiolabeled duplex strand with its complementary biotinylated strand following denaturation. In addition, the cross-links were completely reversible upon irradiation at 254 nm, as expected for psoralen-mediated cross-links. The yield and distribution of photoadducts depended on 5'-TpA position. The most efficient photoadduct formation (approximately 90%) and photo-cross-link formation (approximately 90% of total photoadducts) were observed for a 5'-TpA adjacent to the triplex junction, with significant, but lower, cross-linking efficiency within three base pairs of the junction. Molecular models of the psoralen-conjugated triplex with its six-carbon linker suggested a simple explanation for this distance dependence: facile intercalation near the triplex/duplex junction, with increasing strain required for intercalation at more distant sites. These results indicate that psorTFO1 allows for DNA damage with high precision and high efficiency, and the likely proportion of monoadducts and cross-links can be estimated from the target sequence.

Specific recognition of duplex DNA by a single-stranded oligonucleotide via the formation of triplex DNA is a rational approach for targeting specific regions of a genome. By screening a number of potential target sites for triple helix formation within mammalian genes that allow genetic selection in cell culture, we have identified a site within intron 1 of the hamster adenine phosphoribosyltransferase (APRT) gene that specifically binds a triplex-forming oligodeoxyribonucleotide (TFO) with high affinity. Under optimal conditions for triplex formation, the equilibrium dissociation constant is in the nanomolar range (Kd = 7 x 10(-10) M). This high-affinity binding is very specific, as a 10(5)-fold excess of genomic DNA reduced triplex formation less than 10-fold, and within a 6928-bp plasmid bearing the APRT gene, only restriction fragments containing the intron 1 site were found to bind the TFO. Results of DNase I protection assays were consistent with the TFO binding in an antiparallel orientation via reverse Hoogsteen hydrogen bonds in the major groove of the duplex. We have examined the kinetics of triplex formation as well as the effects of ionic composition and chemical modifications of the TFO on triplex formation. While divalent cations were not required for triplex formation, Mg2+ stabilized the triplex apparently through inhibition of TFO dissociation, with a mean bound lifetime of > 17 h for the triplex at Mg2+ concentrations above 5 mM.(ABSTRACT TRUNCATED AT 250 WORDS)