Shinohara Ken-Ichi, Narita Akihiko, Oyoshi Takanori, Bando Toshikazu, Teraoka Hirobumi, Sugiyama Hiroshi
Department of Pathological Biochemistry, Medical Research Institute, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo 101-0062, Japan.
J Am Chem Soc. 2004 Apr 28;126(16):5113-8. doi: 10.1021/ja031673v.
Gene silencing was examined by sequence-specific alkylation of DNA by N-methylpyrrole (Py)-N-methylimidazole (Im) hairpin polyamides. Polyamides ImImPyPygammaImImPyLDu86 (A) and ImImPyPygammaImPyPyLDu86 (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.
通过N-甲基吡咯（Py）-N-甲基咪唑（Im）发夹型聚酰胺对DNA进行序列特异性烷基化来检测基因沉默。根据Py-Im聚酰胺的碱基对识别规则，聚酰胺ImImPyPygammaImImPyLDu86（A）和ImImPyPygammaImPyPyLDu86（B）分别选择性地烷基化海肾荧光素酶和萤火虫荧光素酶的编码区。使用两种不同的质粒，分别编码海肾荧光素酶和萤火虫荧光素酶，作为载体来检测烷基化对基因沉默的影响。将经聚酰胺A或B烷基化的荧光素酶载体转染到HeLa、293和NIH3T3细胞中，结果表明这些序列特异性DNA烷基化导致基因表达的选择性沉默。接下来，将这些载体共转染到HeLa细胞中，并用聚酰胺A或B处理细胞。两种聚酰胺均导致荧光素酶活性的选择性降低。基于这种序列特异性烷基化和基因沉默活性，这些烷基化的Py-Im聚酰胺因此具有作为抗肿瘤药物靶向人类细胞中特定基因表达的潜力。
