Accessibility of nuclear DNA to triplex-forming oligonucleotides: The integrated HIV-1 provirus as a target (oligonucleotide–psoralen conjugateyDNA accessibilityycompetitive PCRyantigene oligonucleotidesyHIV)

The control of gene transcription by antigene oligonucleotides rests upon the specific recognition of doublehelical 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. Oligonucleotides can be designed to bind to double-stranded DNA at oligopurinezoligopyrimidine sites where they form a local triple-helical structure (see refs. 1 and 2 for reviews). This provides a general approach to the sequence-specific recognition and targeting of double-stranded DNA for both basic research and therapeutic applications. However, the description of triplex-mediated effects in cell cultures is still rare. Inhibition of gene expression by triplex-forming oligonucleotides has been demonstrated on plasmid targets transiently transfected into living cells (3–7). When a triplex was preformed with the target in vitro and then the complex was transfected into mammalian cells, inhibition of gene expression was observed. We have previously shown that transcription of the gene coding for the a-subunit of the interleukin 2 receptor was inhibited when cells were first transfected with a reporter plasmid in the presence of a psoralen– oligonucleotide conjugate and then irradiated (3), or when transfected cells were incubated with an acridine– oligonucleotide conjugate without any irradiation (8, 9). Mutations have been detected on plasmid vectors after irradiation of transfected cells in the presence of psoralen– oligonucleotide conjugates (10). Even in the absence of irradiation a low level of mutation was detected and was attributed to transcription-coupled repair (11). Location of the mutation sites was as it was expected on the basis of triplex formation. Detection of the DNA mutations is a very sensitive method to demonstrate triplex formation, but it does not allow quantitative analysis of the amount of triplex formed within cells. Using dimethyl sulfate footprinting technique, Svinarchuk et al. (7) were unable to detect triplex formation in transfected cells that were further incubated with a triplex-forming oligonucleotide. The footprint was detected only when the complex was preformed in vitro before transfection. There have been several reports describing the use of triplex-forming oligonucleotides to inhibit the expression of endogenous genes (12–17). However, none of these reports provided direct evidence for the implication of triplex formation at the target site in the inhibitory activity. Even when there is an indication of triplex formation [based on inhibition of DNase I hypersensitive sites on the c-myc gene, (13)], the mechanism of gene inhibition more likely involves transcription factor binding to the G-rich oligonucleotide acting as a decoy (18). In the present study we demonstrate that a triplex-forming oligonucleotide, directed against the HIV-1 polypurine tract, can specifically recognize and bind its 15 bp target located on nuclear DNA involved in the intact supranucleosomal structure of chromatin. This is based on a procedure we have developed using an oligonucleotide–psoralen conjugate as a tool to trap the triple-helical complex formed in the cell nucleus. This approach exploits the photochemical reaction of psoralen with the DNA strands at the triplex site which converts a noncovalent triplex into a covalent one and results in a localized damage on genomic DNA. Two different assays were used to quantitatively analyze the oligonucleotide– psoralen cross-links to genomic DNA. These procedures allowed us to examine triplex formation within the cell nuclei directly and to evaluate both the specificity and the efficiency of the reaction. MATERIALS AND METHODS Oligonucleotides. The unmodified oligodeoxynucleotides used as PCR primers in this study were obtained from Eurogentec (Brussels) (see sequences in legend of Fig. 3A). Oligonucleotide analogues with N39 3 P59 phosphoramidate (np) linkages were synthesized as described (19). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA 0027-8424y97y9479-6$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: PPT, polypurine tract; po, phosphodiester; np, phosphoramidate. †To whom reprint requests should be addressed. e-mail: giovanna@ mnhn.fr.