Reversible inhibition of gene expression by a psoralen functionalized triple helix forming oligonucleotide in intact cells.
Triple helix formation of nucleic acids is the most rational approach to designing site-specific transcription inhibitors. To increase their efficiency, reactive moieties such as psoralen or ethenocytosine have been introduced on the third strand. In transfected cells, these compounds induce a site-specific covalent binding of the third strand to the targeted sequence and efficiently block RNA polymerases. However, the stability of this transcription inhibition has never been checked. We have designed a plasmid containing a triple helix binding site in the coding region of the beta-galactosidase reporter gene and a polymerase chain reaction assay to follow quantitatively the cross-link of a psoralen-derivatized third strand in transfected cells. This assay has revealed that the cross-link was removed within a few hours, leading only to a transitory inhibition of gene expression. Control experiments in DNA repair-deficient cells suggest the implication of repair enzymes in this process.
Degols
G
G
Institut de Génétique Móléculaire du CNRS, Montpellier, France.
Clarenc
J P
JP
Lebleu
B
B
Léonetti
J P
JP
D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004260:DNA Repair; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors*; D011564:Furocoumarins / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects*; D005798:Genes, Bacterial; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000494:pharmacology*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D012867:Skin; D014162:Transfection; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D011564:Furocoumarins; D009838:Oligodeoxyribonucleotides; D012321:DNA-Directed RNA Polymerases; D001616:beta-Galactosidase