A SHORT PURINE OLIGONUCLEOTIDE FORMS A HIGHLY STABLE TRIPLE-HELIX WITH THE PROMOTER OF THE MURINE C-PIM-1 PROTOONCOGENE

A homopurine - homopyrimidine region of murine c-pim-1 proto-oncogene was chosen as a target for triple-helix-forming oligonucleotide. Oligonucleotide 5'-GGGGAGGGGGAGG-3' was shown to bind to its target sequence in the presence of 50 mM Na+ or K+, 10 mM MgCl2 and 20 mM Tris-acetate, pH 7.5. This oligonucleotide is bound in an antiparallel orientation with respect to the homopurine sequence. As was shown by co-migration assay the tripler is stable up to 65 degrees C. At 37 degrees C it was practically irreversible: after 24 hours of co-migration assay there was no traces of tripler dissociation. The rate of tripler formation was highly accelerated with increase of temperature and Mg2+ concentration. This rate was higher for superhelical DNA when compared to the linear and circular ones and the preference was dependent from temperature and Mg2+ concentration. The precision of this interaction is extremely high: sequences in c-pim-l promoter region with only one substitution when compared to the target gave negligible tripler formation under investigated conditions. These data suppose that natural tripler structures could play an important role in eukaryotic gene regulation and/or chromatin structure formation.