BINDING OF DNA OLIGONUCLEOTIDES TO SEQUENCES IN THE PROMOTER OF THE HUMAN BCL-2 GENE

Duplex DNA recognition by oligonucleotide-directed triple helix format ion is being explored as a highly specific approach to artificial gene repression. We have identified two potential tripler target sequences in the promoter of the human bcl-2 gene, whose product inhibits apopt osis. Oligonucleotides designed to bind these target sequences were te sted for their binding affinities and specificities under pseudophysio logical conditions. Electrophoretic mobility shift and dimethyl sulfat e footprinting assays demonstrated that an oligonucleotide designed fo r simultaneous recognition of homopurine domains on alternate duplex D NA strands had the highest affinity of any oligonucleotide tested. Mod ifications to render this oligonucleotide nuclease-resistant did not r educe its binding affinity or specificity. In additional studies under various pH conditions, pyrimidine motif complexes at these target seq uences were found to be stable at pH 8.0, despite the presumed require ment for protonation of oligonucleotide cytidines. In contrast, purine motif complexes, typically considered to be pH independent, were high ly destabilized at decreasing pH values. These results indicate that a natural sequence in the human bcl-2 promoter can form a stable triple r with a synthetic oligonucleotide under pseudo-physiological conditio ns, and suggest that triple helix formation might provide an approach to the artificial repression of bcl-2 transcription.