Triplex Formation by Oligonucleotides Containing 5-(1-Propynyl)-2'-deoxyuridine: Decreased Magnesium Dependence and Improved Intracellular Gene Targeting
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- 作者：Laurent Lacroix ; Jé ; rô ; me Lacoste ; James F. Reddoch ; Jean-Louis Mergny ; Dan D. Levy ; Michael M. Seidman ; Mark D. Matteucci ; and Peter M. Glazer
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：February 9, 1999
- 年：1999
- 卷：38
- 期：6
- 页码：1893 - 1901
- 全文大小：147K
- 年卷期：v.38,no.6(February 9, 1999)
- ISSN：1520-4995
文摘
Oligonucleotides capable of sequence-specific triple helix formation have been proposed asDNA binding ligands useful for modulation of gene expression and for directed genome modification.However, the effectiveness of such triplex-forming oligonucleotides (TFOs) depends on their ability tobind to their target sites within cells, and this can be limited under physiologic conditions. In particular,triplex formation in the pyrimidine motif is favored by unphysiologically low pH and high magnesiumconcentrations. To address these limitations, a series of pyrimidine TFOs were tested for third-strandbinding 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 magnesiumconcentrations, as determined by gel mobility shift assays and thermal dissociation profiles. Over a rangeof Mg2+ concentrations, pdU-modified TFOs formed more stable triplexes than did TFOs containing2'-deoxythymidine. At 1 mM Mg2+, a 
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f">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 inmammalian cells, yielding 4-fold higher mutation frequencies in a shuttle vector-based mutagenesis assaydesigned to detect mutations induced by third-strand-directed psoralen adducts. These results suggest theutility of the pdU substitution in the pyrimidine motif for triplex-based gene targeting experiments.
fchars/Delta.gif" BORDER=0 >Tm of 30 f">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 inmammalian cells, yielding 4-fold higher mutation frequencies in a shuttle vector-based mutagenesis assaydesigned to detect mutations induced by third-strand-directed psoralen adducts. These results suggest theutility of the pdU substitution in the pyrimidine motif for triplex-based gene targeting experiments.