Triplex formation by oligonucleotides containing 5-(1-propynyl)-2′-deoxyuridine:: Decreased magnesium dependence and improved intracellular gene targeting
Triplex formation by oligonucleotides containing 5-(1-propynyl)-2′-deoxyuridine:: Decreased magnesium dependence and improved intracellular gene targeting
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DOI：
10.1021/bi982290q
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发表时间：
1999-02-09
期刊：
影响因子：
2.9
通讯作者：
Glazer, PM
中科院分区：
文献类型：
--
作者：
Lacroix, L;Lacoste, J;Glazer, PM
Oligonucleotides capable of sequence-specific triple helix formation have been proposed as DNA binding ligands useful for modulation of gene expression 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 favored by unphysiologically low pH and high magnesium concentrations. To address these limitations, a series of pyrimidine TFOs were tested for third-strand binding under a variety of conditions. Those containing 5-(1-propynyl)-2'-deoxyuridine (pdU) and 5-methyl-2'-deoxycytidine (5meC) showed superior binding characteristics at neutral pH and at low magnesium concentrations, as determined by gel mobility shift assays and thermal dissociation profiles. Over a range of Mg2+ concentrations, pdU-modified TFOs formed more stable triplexes than did TFOs containing 2'-deoxythymidine. At 1 mM Mg2+, a Delta 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 pdU-containing TFOs were superior in gene targeting experiments in mammalian cells, yielding 4-fold higher mutation frequencies in a shuttle vector-based mutagenesis assay designed to detect mutations induced by third-strand-directed psoralen adducts. These results suggest the utility of the pdU substitution in the pyrimidine motif for tripler-based gene targeting experiments.
