Accessibility of nuclear DNA to triplex-forming oligonucleotides: The integrated HIV-1 provirus as a target

Abstract

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 oligopurine⅐oligopyrimidine 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 ␣-subunit of the interleukin 2 receptor was inhibited when cells were first transfected with a reporter plasmid in the presence of a psoralenoligonucleotide conjugate and then irradiated (3), or when transfected cells were incubated with an acridineoligonucleotide conjugate without any irradiation (8, 9). Mutations have been detected on plasmid vectors after irradiation of transfected cells in the presence of psoralenoligonucleotide 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 ...

DNA triple helices: Biological consequences and therapeutic potential

“…Among these are chemical modifications that result in neutral or cationic backbones to reduce the electrostatic repulsion between the negatively charged phosphodiester backbone of the TFO with that of the target DNA duplex. Examples of such modifications include thioate linkages [56,57], N 3' − P 5' phosphoramidates [58,59], and morpholino phosphoramidate linkages [60]. The cationic phosphoramidate linkages, N,Ndiethylethylenediamine and N,N-dimethylaminopropylamine, confer increases in TFO binding affinity and intracellular activity [61].…”

Interactions of cryptolepine and neocryptolepine with unusual DNA structures

“…1B) [10,11]. Triplex-forming oligonucleotides (TFOs) can interfere with the binding of proteins [12] and affect the transcription of a specific gene [13]. At least three classes of triplexes exist which differ in sequence composition and relative orientations of the phosphodiester backbone of the third strand [14].…”

Bioconjugation of Oligonucleotides for Treating Liver Fibrosis

“…When the target sequence was located in transcription region, the triplex formation efficiency varied from one gene to another and was not correlated with the status of gene expression. However, different degrees of triplex formation were observed when HIV-1 genome was integrated into different sites of the host genome (396). Since transcriptional activation can change chromatin organization including nucleosome disruption or displacement in several systems (399,400), the transcription status of target DNA sequences must affect the triplex formation efficiency.…”

“…The accessibility is dependent on the sites of target sequences and TFOs used. In a pioneer study, Giovannangeli et al demonstrated triplex formation of psoralen modified 15-mer PO-or PN-TFOs with genomic DNA within permeabilized cells chronically infected with viral HIV-1 (396). PN-TFO showed higher efficiency in triplex formation than PO analogue.…”

“…This is especially true for the targets in the genome context extracted from nuclei or cells, since the amount of the expected fragmental DNA is extremely low. In one study, the triplex forming sequence was in HIV-1 genome, which was integrated into cellular genome (396). TFOs used were PN analogue with psoralen modification.…”