TY - JOUR
T1 - Binding of DNA oligonucleotides to sequences in the promoter of the human bcl-2 gene
AU - Olivas, Wendy M.
AU - Maher, L. James
N1 - Funding Information:
We gratefully acknowledge D. Eicher and C. Mountjoy for their excellent technical assistance. This work was supported by NIH grant GM 47814 and a Junior Faculty Research Award from The American Cancer Society to L.J.M. W.M.O. is supported by a University of Nebraska Medical Center Presidential Fellowship.
PY - 1996
Y1 - 1996
N2 - 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.
AB - 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.
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U2 - 10.1093/nar/24.9.1758
DO - 10.1093/nar/24.9.1758
M3 - Article
C2 - 8649997
AN - SCOPUS:0029919306
SN - 0305-1048
VL - 24
SP - 1758
EP - 1764
JO - Nucleic acids research
JF - Nucleic acids research
IS - 9
ER -