Inhibition of transcription of the human c-myc protooncogene by intermolecular triplex

Tripler-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;he proliferation of these cells. In an attempt to assay the ability of tripler-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. Tripler 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 tripler formation may represent a gene-specific means of inhibiting specific protooncogene expression.