A bis-alkylating triplex forming oligonucleotide inhibits intracellular reporter gene expression and prevents triplex unwinding due to helicase activity.
A bis-alkylating triplex forming oligonucleotide inhibits intracellular reporter gene expression and prevents triplex unwinding due to helicase activity.
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双烷基化三链体形成寡核苷酸抑制细胞内报告基因表达并防止由于解旋酶活性而导致的三链体解旋。
DOI：
10.1021/bi0273112
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Ebbinghaus,ScotW
中科院分区：
文献类型：
--
作者：
Ziemba,AmyJ;Reed,MichaelW;Raney,KevinD;Byrd,AliciaB;Ebbinghaus,ScotW
三链体形成寡核苷酸 (TFO) 能够通过形成三螺旋 DNA 来位点特异性调节基因表达。 HER-2/neu启动子包含一个战略性定位的三联靶序列，并已在体外成功靶向，但在体内却收效甚微。 TFO 在其 5' 和 3' 末端与烷化剂（苯乙酸芥）缀合，试图稳定细胞内的三螺旋。体外测定表明，双缀合物结合双链体并高效烷基化目标鸟嘌呤残基。使用具有预制三螺旋的质粒，双缀合物将癌细胞中的启动子活性抑制了 60-70%，并且当氮芥仅在一端缀合时，抑制作用最小。解旋酶测定表明，解旋酶活性可以解旋三螺旋未烷基化末端的 TFO，这可能会使解旋的寡核苷酸容易受到核酸酶降解的影响或无法有效抑制转录起始。我们的研究结果表明，需要靶序列的双烷基化来抑制具有预先形成的三螺旋的报告质粒的细胞内活性，这可能是由于细胞内三螺旋的稳定性更高以及解旋酶活性的抑制。
Triplex forming oligonucleotides (TFOs) have the ability to site specifically modulate gene expression through the formation of triple helix DNA. The HER-2/neu promoter contains a strategically located triplex target sequence, and has been successfully targeted in vitro, with little success in vivo. A TFO was conjugated at both its 5‘ and 3‘ ends to an alkylating agent (phenylacetate mustard) in an attempt to stabilize the triple helix intracellularly. In vitro assays demonstrated that the bis-conjugate bound the duplex and alkylated the target guanine residues with high efficiency. The bis-conjugate suppressed promoter activity by 60−70% in cancer cells using a plasmid with a preformed triple helix, and the suppression was minimal when the nitrogen mustard was conjugated at only one end. Helicase assays demonstrated that helicase activity can unwind the TFO at the unalkylated end of the triple helix, which may leave the unwound oligonucleotide susceptible to nuclease degradation or ineffective at inhibiting transcription initiation. Our findings indicate that dual alkylation of the target sequence is required to suppress the intracellular activity of a reporter plasmid with a preformed triple helix, likely due to greater stability of the triple helix within cells and inhibition of helicase activity.