title: Alkylation of template strand of coding region causes effective gene silencing.
abstract: 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.
authors.affiliation: Department of Chemistry, Graduate School of Science, Kyoto University Kitashirakawa-Oiwakecho, Sakyo, Kyoto, 606-8502, Japan.
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Shinohara
authors.forename: Shunta
authors.initials: S
authors.lastname: Sasaki
authors.forename: Masafumi
authors.initials: M
authors.lastname: Minoshima
authors.forename: Toshikazu
authors.initials: T
authors.lastname: Bando
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Sugiyama
mesh_terms: D000478:Alkylation; D045744:Cell Line, Tumor; D004247:DNA / Q000187:drug effects; D020868:Gene Silencing; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006801:Humans; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D009757:Nylons / Q000737:chemistry / Q000494:pharmacology / Q000633:toxicity; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis; D013698:Templates, Genetic
chemical_list: D009757:Nylons; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D004247:DNA