Abstract
Triplex-forming oligonucleotides (TFOs) can bind to polypurine/polypyrimidine regions in DNA in a sequence-specific manner and provoke DNA repair. We have coupled a TFO to a short donor fragment of DNA that shares homology to a selected gene as a strategy to mediate gene targeting and correction. In this bifunctional oligonucleotide, the TFO domain is designed to bind the target gene and stimulate repair and recombination, with the donor domain positioned for recombination and information transfer. A series of these tethered donor-TFO (TD-TFO) molecules with donor domains of 40-44 nucleotides and TFO domains in both the purine and pyrimidine triplex motifs were tested for their ability to mediate either gene correction or mutation of a supF reporter gene contained in a SV40 shuttle vector in mammalian cells. In vitro binding assays revealed that the attachment of the donor domain via a flexible linker did not significantly alter the binding affinity of the TFO domain for the polypurine site in the supF target DNA, with equilibrium dissociation constants in the 10(-8) M range. Experiments in which the target vector and the linked TD-TFOs were pre-incubated in vitro and co-transfected into cells led to conversion frequencies approaching 1%, 4-fold greater than with the two domains unlinked. When cells that had been previously transfected with the SV40 vector were electroporated with the TD-TFOs, frequencies of base pair-specific gene correction were seen in the range of 0.04%, up to 50-fold over background and at least 3-fold over either domain alone or in unlinked combinations. Sequence conversion by the TD-TFOs was achieved using either single- or double-stranded donor domains and either triplex motif. Substitution of either domain in the TD-TFO with control sequences yielded reagents with diminished activity, as did mixtures of unlinked TFO and donor DNA segments. The boost in activity provided by the attached TFO domain was reduced in cells deficient in the nucleotide excision repair factor XPA but was restored in a subclone of these cells expressing XPA cDNA, suggesting a role for nucleotide excision repair in the pathway of triple helix-stimulated gene conversion. The ability to correct or mutate a specific target site in mammalian cells using the TD-TFO strategy may provide a useful tool for research and possibly for therapeutic applications.
Highlights
Introduction of Specific Mutations intoWild-type supFG1— We further tested the activity of TD-triplex-forming oligonucleotides (TFOs) molecules by assaying their ability to introduce a variety of sequence changes into a wild-type supFG1 gene in a forward mutation assay
Using a SV40-based shuttle vector assay, we demonstrate that these TD-TFOs can mediate specific and directed sequence changes within an extrachromosomal supF reporter gene in mammalian cells
Using the protocol described above, we found that A/B(144)-AG30 induced a mutation frequency of 0.64% (Fig. 5B), approximately 9-fold higher than the spontaneous forward mutation frequency of 0.07% and higher than that of the single-stranded A(144)-AG30 (0.30%). (Note that the background frequency in this forward mutation assay is much higher than that seen in the reversion assay because many nonspecific sequence changes can inactivate the gene)
Summary
Vol 274, No 17, Issue of April 23, pp. 11541–11548, 1999 Printed in U.S.A. Targeted Correction of an Episomal Gene in Mammalian Cells by a Short DNA Fragment Tethered to a Triplex-forming Oligonucleotide*. One approach utilizes triplex-forming oligonucleotides (TFOs), which bind as third strands to duplex DNA in a sequence-specific manner, to mediate directed mutagenesis Such TFOs can act either by delivering a tethered mutagen, such as psoralen or chlorambucil [1,2,3,4,5], or by binding with sufficient affinity to provoke error-prone repair [6]. We describe the design of several TD-TFO reagents These bind and with high affinity to DNA via a triplex-forming domain while simultaneously providing DNA sequence information through an attached donor domain to revert or induce a mutation in a target gene (Fig. 1). Using human mutant cell lines, we demonstrate that the enhanced activity of the combined TD-TFO is diminished in cells deficient in nucleotide excision repair (NER), suggesting that the TFO domain may stimulate gene conversion in part through the ability of triple helices to provoke repair
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