INHIBITION OF GENE-EXPRESSION BY TRIPLE HELIX-DIRECTED DNA CROSS-LINKING AT SPECIFIC SITES

Synthetic oligodeoxynucleotides represent promising tools for gene inh ibition in live systems. Triple helix-forming oligonucleotides, which bind to double-stranded DNA, are of special interest since they are ta rgeted to the gene itself rather than to its mRNA product, as in the a ntisense strategy. Triple helix-forming oligonucleotides can be couple d to DNA-modifying agents and used to introduce modifications in the D NA target in a highly sequence-specific manner. We have recently desig ned psoralen-oligonucleotide conjugates, which, upon binding to double -stranded DNA sequences via triple helix formation, may be cross-linke d in vitro to both strands of the DNA following UV irradiation. A psor alen-oligonucleotide conjugate was targeted to the promoter of the alp ha subunit of the interleukin 2 receptor (IL-2Ralpaha) gene. The tripl e helix site overlaps the binding site for the transcription factor NF -kappaB, which activates transcription from the IL-2Ralpha promoter. A fter UV irradiation, the oligonucleotide conjugate becomes crosslinked to the target site and inhibits transcription of reporter plasmids tr ansfected in live cells. Inhibition is observed when UV-induced cross- linking occurs both in vitro (before transfection) and in vivo (after transfection). We directly demonstrate that this inhibitory effect is due to triple helix formation at the target site, since a mutant of th e promoter, to which oligonucleotide binding was inhibited, was not af fected by the psoralen-oligonucleotide conjugate after UV irradiation. In addition, we demonstrate that site-specific cross-linking upstream of the promoter has no effect on transcription.