INHIBITION OF IN-VITRO TRANSCRIPTION BY A TRIPLEX-FORMING OLIGONUCLEOTIDE TARGETED TO HUMAN C-MYC P2 PROMOTER

Tripler-forming oligonucleotides (TFOs) have been shown to bind in a sequence-specific manner to polypurine/polypyrimidine sequences in several human gene promoters, including the c-myc P1 promoter. TFOs have been shown to inhibit transcription in vitro and the expression of target genes in cell culture. The human c-myc protooncogene contains a 23 base pair purine-pyrimidine-rich motif (-62 to -40) within its predominant promoter, P2, that is a potential target for purine-purine-pyrimidine tripler formation. Using electrophoretic mobility shift analysis (EMSA) and competition experiments, we have demonstrated that a MAZ (myc-associated zinc finger protein) consensus sequence is capable of competing with the purine-pyrimidine motif for the binding of a HeLa nuclear protein. We have shown the formation of an intermolecular tripler using a 23-base purine-rich oligonucleotide antiparallel to the purine-rich target sequence, DNase I footprinting was performed to confirm the exact location of tripler formation. Tripler formation by this oligonucleotide prevents binding of a HeLa nuclear protein (presumably MAZ) to the target site. We have also shown that the P2-targeted TFO is a potent and specific inhibitor of c-myc transcription in vitro. These data demonstrate that this novel TFO inhibits transcription of the c-myc P2 promoter. We propose that the P2-targeted TFO has its effect by blocking the binding of the regulatory factor MAZ.