We are developing a gene targeting program based on oligonucleotides that form stable triple helix complexes with specific sequences in duplex DNA. This approach has the promise to become a simple and efficient technology for delivering DNA reactive compounds to specific sites in chromosomal DNA in living cells. Applications include gene knockout, directed gene conversion and recombination, and, perhaps, gene therapy. We have prepared TFOs containing novel sugar analogues and have identified a modification format that supports efficient targeting of specific chromosomal sequences in living mammalian cells. In our developmental work we prepared TFOs, linked to a photoactive DNA mutagen, directed against a sequence in a gene (HPRT) frequently used as a mutation reporter. We introduced this into mammalian cells and, after photoactivation of the mutagen, isolated colonies of cells with mutations in the target gene. Sequence analysis showed that the mutations were located at the target sequence within the gene. Treatment of S phase cells with these TFOs resulted in targeted crosslinking in 30% of the cells and 5-10% mutation frequencies. Both crosslinking and mutagenesis were much lower in quiescent cells. These results indicate that the accessibility of chromosomal target sites in mammalian cells is modulated by the biology of the cell. This strategy has been extended to other genes for which genetic analyses of targeting are not possible. For example, we have recently shown, using a biochemical assay, that a site in the human beta globin gene can be targeted at high efficiency. Additional analyses of the oligonucleotide chemistry required for bioactivity revealed that the active TFOs contain a balance of modifications, too much or too little reduces activity. Triplex formation is most stable on polypurine:polypyrimidine sequences, and TFOs against targets with interruptions in this arrangement are generally not successful. We have synthesized a novel base analogue that permits stable triplex formation on a target sequence containing a C:G interruption in the polypurine:polypyrimidine element. We have shown that TFOs with this analogue form stable triplexes in vitro. We constructed a novel cell line by targeted sequence conversion in which the standard polypurine:polypyrimidine target was replaced with a target containing the C:G interruption. We then treated the standard line and the isogenic line cell line with the new TFO and the standard TFO. The standard TFO was effective against the cells with the standard target while the TFO with the novel analogue was active against the cells with the interrupted target but not the standard target. This is the first demonstration of biological activity of a base analogue that enables a TFO to overcome the limitations on target sequence. The results of molecular modeling of the novel and standard triplex structures are consistent with the biochemical and biological data.
