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It began as a curious observation by physical chemists in 1957: a double-stranded DNA can bind another short molecule of DNA to form a triple helix. However, not until the synthesis of short DNA oligonucleotides became routine did this finding capture the imaginations of biologists; now it became possible to make TFOs that targeted specific genomic sequences and subsequently study the effect of this triplex DNA on, for example, transcription, mutagenesis or recombination.

Peter Glazer at Yale University has long been interested in the ability of TFOs to induce mutations and initiate DNA recombination. However, his and other researchers' studies were hampered by the fact that TFOs are not easily taken up by the cell. Glazer realized that without the ability to efficiently deliver these molecules to the nucleus of a cell, their use would be very limited. A recent article in Nucleic Acids Research describes his method to improve nuclear TFO delivery (Rogers et al., 2004).

His team started with a translocation peptide, previously known to transport molecules into the cytosol of a cell and covalently linked it to a TFO. They found that the mutation frequency in the targeted gene in cells treated with the TFO-peptide hybrid was 20 times higher than in cells treated with the TFO alone. Glazer attributes this effect to the dual function of the peptides: “They shuttle the attached DNA through the plasma membrane into the cell and then into the nucleus. Their positive charge helps neutralize the negative charge of the DNA double helix and so aids the binding of the TFO to form the triplex.”

By adding a nuclear localization signal to the translocation peptide, Glazer hopes to improve the nuclear delivery of TFO-peptide hybrids even more. This improvement in the efficiency of uptake is likely to usher in a bright future for TFOs in applications such as targeted gene knock out.