Accessibility of nuclear DNA to triplex-forming oligonucleotides: the integrated HIV-1 provirus as a target.
The control of gene transcription by antigene oligonucleotides rests upon the specific recognition of double-helical DNA by triplex-forming oligonucleotides. The development of the antigene strategy requires access to the targeted DNA sequence within the chromatin structure of the cell nucleus. In this sudy we have used HIV-1 chronically infected cells containing the HIV provirus as endogenous genes to demonstrate that the integrated HIV-1 proviral genome is accessible to triplex-forming oligonucleotides within cell nuclei. An oligonucleotide-psoralen conjugate targeted to the polypurine tract (PPT) of the HIV-1 proviral sequence was used as a tool to convert the noncovalent triple-helical complex into a covalent lesion on genomic DNA after UV irradiation of cells. Triplex-derived adducts were analyzed using two different methods. The photo-induced psoralen cross-link prevented cleavage of the target sequence by DraI restriction endonuclease, and the sequence-specific inhibition of cleavage was revealed and quantitated by Southern blot analysis. A quantitative analysis of cross-linking efficiency was also carried out by a competitive PCR-based assay. These two approaches allowed us to demonstrate that a triplex-forming oligonucleotide can recognize and bind specifically to a 15-bp sequence within the chromatin structure of cell nuclei.
Giovannangeli
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Institut National de la Santé et de la Recherche Médicale-Unité 201,Centre National de la Recherche Scientifique, Paris, France. giovanna@mnhn.fr
Diviacco
S
S
Labrousse
V
V
Gryaznov
S
S
Charneau
P
P
Helene
C
C
D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D002843:Chromatin / Q000187:drug effects*; D003432:Cross-Linking Reagents; D004247:DNA / Q000187:drug effects*; D004279:DNA, Viral / Q000187:drug effects*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005363:Ficusin / Q000737:chemistry; D015497:HIV-1 / Q000187:drug effects*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D011533:Proviruses / Q000187:drug effects*
D002843:Chromatin; D003432:Cross-Linking Reagents; D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; C051802:triplex DNA; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C035594:TTTAAA -specific type II deoxyribonucleases; D005363:Ficusin