Alkylation of template strand of coding region causes effective gene silencing
Alkylation of template strand of coding region causes effective gene silencing
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DOI：
10.1093/nar/gkl005
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发表时间：
2006-01-01
影响因子：
14.9
通讯作者：
Sugiyama, H
中科院分区：
文献类型：
--
作者：
Shinohara, K;Sasaki, S;Sugiyama, H
我们最近开发了一种新型的吡咯（Py）-咪唑（Im）聚酰胺-四氢环丙烷苯并吲哚酮（CBI）共轭物，它带有一个吲哚接头作为一种稳定的序列特异性烷基化剂。在这项研究中，我们研究了聚酰胺A，B和C的基因沉默活性，它们分别选择性地烷基化绿色荧光蛋白（GFP）基因的启动子区，非编码链和编码链中的特定序列。将GFP载体转染到人结肠癌细胞（HCT 116）中，并用100 nM聚酰胺处理细胞24小时。荧光显微镜显示，GFP荧光的显著减少仅在用聚酰胺C处理的细胞中观察到。与此形成鲜明对比的是，聚酰胺A和B没有显示出这种活性。此外，实时PCR证实了用聚酰胺C处理后GFP mRNA的表达的选择性降低。这些结果表明，靶向编码链的烷基化Py-Im聚酰胺代表了序列特异性基因沉默的新方法。
We recently developed a new type of pyrrole (Py)-imidazole (Im) polyamide-tetrahydrocyclopropabenzindolone (CBI) conjugate with an indole linker as a stable sequence-specific alkylating agent. In this study, we investigated the gene silencing activities of polyamides A, B and C, which selectively alkylate specific sequences in the promoter region, non-coding strand and coding strand, respectively, of the green fluorescent protein (GFP) gene. GFP vectors were transfected into human colon carcinoma cells (HCT116), and the cells were treated with 100 nM of the polyamides for 24 h. Fluorescence microscopy indicated that a significant reduction of GFP fluorescence was only observed in the cells that were treated with polyamide C. In clear contrast, polyamides A and B did not show such activity. Moreover, real-time PCR demonstrated selective reduction of the expression of GFP mRNA following treatment with polyamide C. These results suggest that alkylating Py-Im polyamides that target the coding strand represent a novel approach for sequence-specific gene silencing.