Sequence-specific gene silencing in mammalian cells by alkylating pyrrole-imidazole polyamides
Sequence-specific gene silencing in mammalian cells by alkylating pyrrole-imidazole polyamides
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DOI：
10.1021/ja031673v
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发表时间：
2004-04-28
影响因子：
15
通讯作者：
Sugiyama, H
中科院分区：
文献类型：
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作者：
Shinohara, K;Narita, A;Sugiyama, H
通过N-甲基吡咯（Py）N-甲基咪唑（Im）发夹聚酰胺对DNA的序列特异性烷基化来检查基因沉默。聚酰胺ImImPyPygammalmPyLDu 86（A）和ImImPyPygammalmPyLDu 86（B）根据Py-Im聚酰胺的碱基对识别规则分别选择性地烷基化海肾和萤火虫酶的编码区。两种不同的质粒，编码海肾荧光素酶和萤火虫荧光素酶，被用作载体，以检查烷基化对基因沉默的影响。通过聚酰胺A或B将烷基化荧光素酶载体转染到HeLa、293和NIH 3 T3细胞中，证明这些序列特异性DNA烷基化导致基因表达的选择性沉默。接着，将载体共转染到HeLa细胞中，并用聚酰胺A或B处理细胞。荧光素酶活性的选择性降低是由两种聚酰胺引起的。基于这种序列特异性烷基化和基因沉默活性，这些烷基化Py-Im聚酰胺因此具有作为靶向人类细胞中特定基因表达的抗肿瘤药物的潜力。
Gene silencing was examined by sequence-specific alkylation of DNA by N-methylpyrrole (Py)N-methylimidazole (Im) hairpin polyamides. Polyamides ImImPyPygammalmlmPyLDu86 (A) and ImImPyPygammalmPyPyLDu86 (B) selectively alkylated the coding regions of the renilla and firefly luciferases, respectively, according to the base pair recognition rule of Py-Im polyamides. Two different plasmids, encoding renilla luciferase and firefly luciferase, were used as vectors to examine the effect of alkylation on gene silencing. Transfection of the alkylated luciferase vectors-by polyamide A or B-into HeLa, 293, and NIH3T3 cells demonstrated that these sequence-specific DNA alkylations lead to selective silencing of gene expression. Next, the vectors were cotransfected into HeLa cells and the cells were treated with polyamide A or B. Selective reduction of luciferase activities was caused by both polyamides. On the basis of this sequence-specific alkylation and gene silencing activity, these alkylating Py-Im polyamides thus have potential as antitumor drugs to target specific gene expression in human cells.