Inhibition of Nuclear Protein Binding to the Human Ki-ras Promoter by Triplex-Forming Oligonucleotides

Abstract

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.