Inhibition of transcription elongation in the HER-2/neu coding sequence by triplex-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 presence and absence of triplex formation demonstrates that an unmodified DNA triplex-forming oligonucleotide is incapable of inhibiting RNA polymerase elongation. Triplex formation by an oligonucleotide-psoralen conjugate was used 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 of the triplex-forming oligonucleotide to the nontemplate strand did not prevent RNA polymerase elongation. Using HER-2/neu point mutants that would place the target thymine on the template strand, we demonstrated that covalent modification of the template strand was necessary to inhibit RNA polymerase elongation. Based on these data, we synthesized oligonucleotide-alkylator conjugates that would react with a specific guanine residue on the template strand of the HER-2/neu coding sequence. The oligonucleotide-alkylator conjugates inhibited transcription elongation by T7 RNA polymerase and eukaryotic RNA polymerase II from a HeLa nuclear extract. These studies demonstrate the successful application of triplex-forming oligonucleotide-alkylator conjugates to inhibit transcription elongation in the HER-2/neu gene, and show that covalent modification of the DNA strand used as the transcription template is necessary to prevent RNA polymerase elongation.
Ebbinghaus
S W
SW
Division of Hematology-Oncology, Department of Medicine, University of Alabama at Birmingham 35295, USA. scot.ebbinghaus@ccc.uab.edu
Fortinberry
H
H
Gamper
H B
HB
D001665:Binding Sites; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018390:Gene Targeting; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D016366:Open Reading Frames / Q000235:genetics*; D010441:Peptide Chain Elongation, Translational / Q000235:genetics*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics*; D013698:Templates, Genetic; D014158:Transcription, Genetic
D004247:DNA; D009838:Oligodeoxyribonucleotides; D018719:Erb-b2 Receptor Tyrosine Kinases; C051802:triplex DNA