Unambiguous demonstration of triple-helix-directed gene modification

Triple-helix-forming oligonucleotides (TFOs), which can potentially modify target genes irreversibly, represent promising tools for antiviral therapie s. However, their effectiveness on endogenous genes has yet to be unambiguo usly demonstrated. To monitor endogenous gene modification by TFOs in a yea st model, we inactivated an auxotrophic marker gene by inserting target seq uences of interest into its coding region, The genetically engineered yeast cells then were treated with psoralen-linked TFOs followed by UV irradiati on, thus generating highly mutagenic covalent crosslinks at the target site whose repair could restore gene function; the number of revertants and spe ctrum of mutations generated were quantified. Results showed that a phospho ramidate TFO indeed reaches its target sequence, forms crosslinks, and gene rates mutations at the expected site via a tripler-mediated mechanism: (i) under identical conditions, no mutations were generated by the same TFO at two other loci in the target strain, nor in an isogenic control strain carr ying a modified target sequence incapable of supporting triple-helix format ion; (ii) for a given target sequence, whether the triplex was formed in vi vo on an endogenous gene or in vitro on an exogenous plasmid, the nature of the mutations generated was identical, and consistent with the repair of a psoralen crosslink at the target site. Although the mutation efficiency wa s probably too low for therapeutic applications, our results confirm the va lidity of the triple-helix approach and provide a means of evaluating the e ffectiveness of new chemically modified TFOs and analogs.