Binding of DNA oligonucleotides to sequences in the promoter of the human bcl-2 gene

Wendy M. Olivas, L. James Maher

Research output: Contribution to journalArticle

16 Scopus citations

Abstract

Duplex DNA recognition by oligonucleotide-directed triple helix formation is being explored as a highly specific approach to artificial gene repression. We have identified two potential triplex target sequences in the promoter of the human bcl-2 gene, whose product inhibits apoptosis. Oligonucleotides designed to bind these target sequences were tested for their binding affinities and specificities under pseudophysiological conditions. Electrophoretic mobility shift and dimethyl sulfate footprinting assays demonstrated that an oligonucleotide designed for simultaneous recognition of homopurine domains on alternate duplex DNA strands had the highest affinity of any oligonucleotide tested. Modifications to render this oligonucleotide nuclease-resistant did not reduce its binding affinity or specificity. In additional studies under various pH conditions, pyrimidine motif complexes at these target sequences were found to be stable at pH 8.0, despite the presumed requirement for protonation of oligonucleotide cytidines. In contrast, purine motif complexes, typically considered to be pH independent, were highly destabilized at decreasing pH values. These results indicate that a natural sequence in the human bcl-2 promoter can form a stable triplex with a synthetic oligonucleotide under pseudo-physiological conditions, and suggest that triple helix formation might provide an approach to the artificial repression of bcl-2 transcription.

Original languageEnglish (US)
Pages (from-to)1758-1764
Number of pages7
JournalNucleic acids research
Volume24
Issue number9
DOIs
StatePublished - May 20 1996

    Fingerprint

ASJC Scopus subject areas

  • Genetics

Cite this