Triplex formation by oligonucleotides containing 5-(1-propynyl)-2 '-deoxyuridine: Decreased magnesium dependence and improved intracellular gene targeting

Oligonucleotides capable of sequence-specific triple helix formation have b een proposed as DNA binding ligands useful for modulation of gene expressio n and for directed genome modification. However, the effectiveness of such tripler-forming oligonucleotides (TFOs) depends on their ability to bind to their target sites within cells, and this can be limited under physiologic conditions. In particular, tripler formation in the pyrimidine motif is fa vored by unphysiologically low pH and high magnesium concentrations. To add ress these limitations, a series of pyrimidine TFOs were tested for third-s trand binding under a variety of conditions. Those containing 5-(1-propynyl )-2'-deoxyuridine (pdU) and 5-methyl-2'-deoxycytidine (5meC) showed superio r binding characteristics at neutral pH and at low magnesium concentrations , as determined by gel mobility shift assays and thermal dissociation profi les. Over a range of Mg2+ concentrations, pdU-modified TFOs formed more sta ble triplexes than did TFOs containing 2'-deoxythymidine. At 1 mM Mg2+, a D elta Tm of 30 degrees C was observed for pdU- versus T-containing 15-mers ( of generic sequence 5' TTTTCTTTTTTCTTTTCT 3') binding to the cognate A:T bp rich site, indicating that pdU-containing TFOs are capable of substantial binding even at physiologically low Mg2+ concentrations. In addition, the p dU-containing TFOs were superior in gene targeting experiments in mammalian cells, yielding 4-fold higher mutation frequencies in a shuttle vector-bas ed mutagenesis assay designed to detect mutations induced by third-strand-d irected psoralen adducts. These results suggest the utility of the pdU subs titution in the pyrimidine motif for tripler-based gene targeting experimen ts.