c‐ fos Protooncogene Transcription can be Modulated by Oligonucleotide‐Mediated Formation of Triplex Structures in vitro

A homopurine · homopyrimidine sequence of the c‐ fos promoter was chosen as a target for a triple helix oligonucleotide. Eight DNA oligonucleotides that ranged from 14 to 31 bp were shown to form a triple helix with three sequences within the c‐ fos promoter region. Reactive derivatives of homopyrimidine oligonucleotides bearing the 5′‐or 3′‐terminal DNA alkylation aromatic 2‐chloroethylamino group were also synthesized. It was concluded, based on the physical properties of the DNA oligonucleotide complex, that the oligonucleotide forms a colinear triplex with the duplex binding sites. We investigated in detail, using electrophoretic mobility and footprinting protection, whether such oligonucleotide · DNA complexes are of benefit in designing high‐affinity probes for a natural DNA sequence in the mouse c‐ fos gene. Our results demonstrate that four different DNA targets within the c‐fos promoter region can form triplex structures with synthetic oligonucleotides in a sequence‐specific manner. Moreover, in vitro modifications of the retinoblastomagene‐product‐binding site of the c‐ fos promoter at position–83 in front of the cAMP/cAMP‐responsive element binding site and fos ‐binding site 3/activator‐protein‐2‐like (FBS3/AP‐2‐like) site at position–431 by triple helix forming oligonucleotides cause dramatic suppression of fos ‐chloramphenicol acetyltransferase activity in endothelial cells. These results provide a basis for the development of a specific oligonucleotide target forming triplex‐DNA complex, and emphasize the importance of a target forming triplex as a basis for control of gene expression and cell proliferation.