Targeting and regulation of the HER-2/neu oncogene promoter with bis-peptide nucleic acids.
Targeting and regulation of the HER-2/neu oncogene promoter with bis-peptide nucleic acids.
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DOI：
10.1089/oli.2005.15.36
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发表时间：
2005-03
期刊：
影响因子：
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通讯作者：
A. Ziemba;Zhanna V. Zhilina;Yulia Krotova-Khan;L. Staňková;S. Ebbinghaus
中科院分区：
文献类型：
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作者：
A. Ziemba;Zhanna V. Zhilina;Yulia Krotova-Khan;L. Staňková;S. Ebbinghaus
抗基因寡核苷酸具有通过位点特异性DNA结合来调节基因表达的潜力。然而，在体内的应用已经受到了低效的细胞摄取，降解和链置换。肽核酸（PNA）解决了这些问题中的几个，因为它们对降解具有抗性并以高亲和力结合DNA。我们设计了两种阳离子嘧啶双PNA（cpy-PNA）靶向HER-2/neu启动子的多嘌呤区，并将其与未修饰的磷酸二酯三链体形成寡核苷酸（TFO 1）和TFO-氮芥缀合物（TFO 2）进行了比较。PNA1含有+2电荷，仅在低pH下以高亲和力和特异性结合两个相邻的9-bp靶序列。PNA2含有+5电荷，在高达pH 7.4下以高亲和力结合一个11-bp靶序列，但特异性较低。由这些cpy-bis-PNA形成的PNA：DNA：PNA三链体对DNA聚合酶延伸呈现稳定的屏障。cpy-双-PNA和TF0-烷基化剂缀合物在报告基因测定中阻止HER-2/neu转录（TF0 2 = PNA1> PNA2>> TF0 1）。PNA和TFO都能有效地结合裸基因组DNA中的靶序列，但在透化细胞中的内源性HER-2/neu启动子处仅检测到TFO-烷基化剂（TFO 2）和阳离子性更强的PNA（PNA 2）。这项工作证明了在肿瘤细胞中用cpy-bis-PNAs预防HER-2/neu基因表达的潜力。
Antigene oligonucleotides have the potential to regulate gene expression through site-specific DNA binding. However, in vivo applications have been hindered by inefficient cellular uptake, degradation, and strand displacement. Peptide nucleic acids (PNAs) address several of these problems, as they are resistant to degradation and bind DNA with high affinity. We designed two cationic pyrimidine bis-PNAs (cpy-PNAs) to target the polypurine tract of the HER-2/neu promoter and compared them to an unmodified phosphodiester triplex-forming oligonucleotide (TFO1) and a TFO-nitrogen mustard conjugate (TFO2). PNA1 contains a + 2 charge and bound two adjacent 9-bp target sequences with high affinity and specificity, but only at low pH. PNA2 contains a +5 charge and bound one 11-bp target with high affinity up to pH 7.4, but with lower specificity. The PNA:DNA:PNA triplex formed by these cpy-bis-PNAs presented a stable barrier to DNA polymerase extension. The cpy-bis-PNAs and the TFO-alkylator conjugate prevented HER-2/neu transcription in a reporter gene assay (TFO2 = PNA1 > PNA2 >> TFO1). Both PNAs and TFOs were effective at binding the target sequence in naked genomic DNA, but only the TFO-alkylator (TFO2) and the more cationic PNA (PNA2) were detected at the endogenous HER-2/neu promoter in permeabilized cells. This work demonstrates the potential for preventing HER-2/neu gene expression with cpy-bis-PNAs in tumor cells.