Inhibition of transcription elongation in the HER-2/neu coding sequence bytriplex-directed covalent modification of the template strand

Triplex formation may be of potential utility to inhibit the expression of individual genes. We describe the formation of a triple helix in the coding sequence of the HER-2/neu gene. In vitro transcription analysis in the pre sence and absence of tripler formation demonstrates that an unmodified DNA triplex-forming oligonucleotide is incapable of inhibiting RNA polymerase e longation. Tripler formation by an oligonucleotide-psoralen conjugate was u sed to form a covalent photoadduct with a thymine on the nontemplate strand of the HER-2/neu gene. In the native HER-2/neu gene, covalent attachment o f the tripler-forming oligonucleotide to the nontemplate strand did not pre vent RNA polymerase elongation. Using HER-2/neu point mutants that would pl ace the target thymine on the template strand, we demonstrated that covalen t modification of the template strand was necessary to inhibit RNA polymera se elongation. Based on these data, we synthesized oligonucleotide-alkylato r conjugates that would react with a specific guanine residue on the templa te strand of the HER-2/neu coding sequence. The oligo nucleotide-alkylator conjugates inhibited transcription elongation by T7 RNA polymerase and euka ryotic RNA polymerase II from a HeLa nuclear extract. These studies demonst rate the successful application of tripler-forming oligonucleotide-alkylato r conjugates to inhibit transcription elongation in the HER-2/neu gene, and show that covalent modification of the DNA strand used as the transcriptio n template is necessary to prevent RNA polymerase elongation.