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 oligonucleot ides that ranged from 14 to 31 bp were shown to form a triple helix wi th three sequences within the c-fos promoter region, Reactive derivati ves 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 oligon ucleotide complex, that the oligonucleotide forms a colinear tripler w ith the duplex binding sites. We investigated in detail, using electro phoretic mobility and footprinting protection, whether such oligonucle otide DNA complexes are of benefit in designing high-affinity probes f or a natural DNA sequence in the mouse c-fos gene. Our results demonst rate that four different DNA targets within the c-Sos promoter region can form tripler structures with synthetic oligonucleotides in a seque nce-specific manner. Moreover, in vitro modifications of thr retinobla stoma-gene-product-binding site of the c-fos promoter at position -83 in front of the cAMP/cAMP-responsive element binding site and fos-bind ing site 3/activator-protein-2-like (FBS3/AP-2-like) site at position -431 by triple helix forming oligonucleotides cause dramatic suppressi on of fos-chloramphenicol acetyltransferase activity in endothelial ce lls. These results provide a basis for the development of a specific o ligonucleotide target forming triplex-DNA complex, and emphasize the i mportance of a target forming triplex as a basis for control of gene e xpression and cell proliferation.