Inhibition of gene expression by triple helix-directed DNA cross-linking at specific sites.
Synthetic oligodeoxynucleotides represent promising tools for gene inhibition in live systems. Triple helix-forming oligonucleotides, which bind to double-stranded DNA, are of special interest since they are targeted to the gene itself rather than to its mRNA product, as in the antisense strategy. Triple helix-forming oligonucleotides can be coupled to DNA-modifying agents and used to introduce modifications in the DNA target in a highly sequence-specific manner. We have recently designed psoralen-oligonucleotide conjugates, which, upon binding to double-stranded DNA sequences via triple helix formation, may be cross-linked in vitro to both strands of the DNA following UV irradiation. A psoralen-oligonucleotide conjugate was targeted to the promoter of the alpha subunit of the interleukin 2 receptor (IL-2R alpha) gene. The triple helix site overlaps the binding site for the transcription factor NF-kappa B, which activates transcription from the IL-2R alpha promoter. After UV irradiation, the oligonucleotide conjugate becomes cross-linked to the target site and inhibits transcription of reporter plasmids transfected 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 the promoter, to which oligonucleotide binding was inhibited, was not affected 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.
Grigoriev
M
M
Laboratoire de Biologie des Tumeurs Humaines, Centre National de la Recherche Scientifique, Villejuif, France.
Praseuth
D
D
Guieysse
A L
AL
Robin
P
P
Thuong
N T
NT
Hélène
C
C
Harel-Bellan
A
A
D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D004247:DNA / Q000235:genetics; D011564:Furocoumarins / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects / Q000528:radiation effects; D016762:Genes, fos / Q000187:drug effects / Q000528:radiation effects; D016325:HIV Long Terminal Repeat; D015368:Human T-lymphotropic virus 1 / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000378:metabolism; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000494:pharmacology*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D015375:Receptors, Interleukin-2 / Q000235:genetics*; D015183:Restriction Mapping; D014158:Transcription, Genetic / Q000187:drug effects* / Q000528:radiation effects; D014162:Transfection; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D011564:Furocoumarins; D016328:NF-kappa B; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D015375:Receptors, Interleukin-2; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase