Kim H G, Reddoch J F, Mayfield C, Ebbinghaus S, Vigneswaran N, Thomas S, Jones D E, Miller D M
Department of Biochemistry, University of Alabama at Birmingham, Birmingham, Alabama 35294-0001, USA.
Biochemistry. 1998 Feb 24;37(8):2299-304. doi: 10.1021/bi9718191.
Triplex-forming oligonucleotides (TFOs) have been shown to inhibit both transcription in vitro and the expression of target genes in cell culture by binding to polypurine/polypyrimidine sequences in several human gene promoters. The c-myc protooncogene is overexpressed in a variety of human cancers and appears to play an important role in the proliferation of these cells. In an attempt to assay the ability of triplex-forming oligonucleotides to inhibit expression of a target gene in vivo, we have developed a cellular system involving transfection of a c-myc promoter-driven luciferase reporter plasmid with triplex-forming oligonucleotides targeted to the human c-myc protooncogene. To increase the stability of the TFO, we have used modified phosphorothioate oligonucleotides. Triplex formation with a modified phosphorothioate oligonucleotide occurs with approximately equal binding affinity as that seen using a phosphodiester oligonucleotide. Phosphorothioate-modified TFOs targeted to c-myc inhibit transcription of the c-myc promoter in HeLa cells as demonstrated by a decrease in luciferase expression from a luciferase reporter gene construct. These results suggests that triplex formation may represent a gene-specific means of inhibiting specific protooncogene expression.
三链形成寡核苷酸（TFOs）已被证明可通过与几种人类基因启动子中的聚嘌呤/聚嘧啶序列结合，在体外抑制转录并在细胞培养中抑制靶基因的表达。c-myc原癌基因在多种人类癌症中过度表达，并且似乎在这些细胞的增殖中起重要作用。为了测定三链形成寡核苷酸在体内抑制靶基因表达的能力，我们开发了一种细胞系统，该系统涉及用靶向人类c-myc原癌基因的三链形成寡核苷酸转染c-myc启动子驱动的荧光素酶报告质粒。为了提高TFO的稳定性，我们使用了修饰的硫代磷酸酯寡核苷酸。与修饰的硫代磷酸酯寡核苷酸形成三链的结合亲和力与使用磷酸二酯寡核苷酸时观察到的大致相同。如荧光素酶报告基因构建体的荧光素酶表达降低所示，靶向c-myc的硫代磷酸酯修饰的TFOs抑制HeLa细胞中c-myc启动子的转录。这些结果表明三链形成可能代表一种抑制特定原癌基因表达的基因特异性方法。
