Inhibition of nuclear protein binding to the human Ki-ras promoter by triplex-forming oligonucleotides.
The human Ki-ras promoter contains a 22 base pair homopurine.homopyrimidine (pur.pyr) motif within a region that is nuclease-hypersensitive in both native chromatin and supercoiled plasmids. Gel mobility shift analysis and competition experiments show that this pur.pyr motif binds a nuclear protein(s) in a sequence-specific manner. Several observations suggest that the nuclear protein may be an important regulatory factor. Gel mobility shift analysis and DNase I footprinting demonstrate that oligonucleotides can be targeted to this motif forming sequence-specific purine*purine.pyrimidine (pur*pur.pyr) or mixed purine/pyrimidine*purine-pyrimidine (pur/pyr*pur.pyr) intermolecular triple helices through guanine (G) recognition of guanine.cytosine (G.C) base pairs and either adenine (A) or thymine (T) recognition of adenine-thymine (A.T) base pairs in the target sequence. Triple helices containing either T*A.T or A*A.T triplets are formed exclusively with oligonucleotides antiparallel to the homopurine target strand. The affinity of an oligonucleotide which forms T*A.T triplets is approximately equal to, or slightly greater than, the affinity of an oligonucleotide which forms A*A.T triplets. Oligonucleotide-directed triplex formation inhibits sequence-specific nuclear protein binding to the K-ras promoter. These observations suggest that triplex formation by the oligonucleotides described here may provide a means to specifically inhibit transcription of the K-ras oncogene.
Mayfield
C
C
Bolden Laboratory, Department of Medicine, University of Alabama at Birmingham 35294-3300.
Squibb
M
M
Miller
D
D
D001482:Base Composition; D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D003850:Deoxyribonuclease I; D011905:Genes, ras; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*
D009687:Nuclear Proteins; D009841:Oligonucleotides; D004247:DNA; D003850:Deoxyribonuclease I; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)