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 purinepurine.pyrimidine (purpur.pyr) or mixed purine/pyrimidinepurine-pyrimidine (pur/pyrpur.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 TA.T or AA.T triplets are formed exclusively with oligonucleotides antiparallel to the homopurine target strand. The affinity of an oligonucleotide which forms TA.T triplets is approximately equal to, or slightly greater than, the affinity of an oligonucleotide which forms AA.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.