Targeting chromosomal sites with locked nucleic acid-modified triplex-forming oligonucleotides: study of efficiency dependence on DNA nuclear environment

Abstract

Triplex-forming oligonucleotides (TFOs) are synthetic DNA code-reading molecules that have been demonstrated to function to some extent in chromatin within cell nuclei. Here we have investigated the impact of DNA nuclear environment on the efficiency of TFO binding. For this study we have used locked nucleic acid-containing TFOs (TFO/LNAs) and we report the development of a rapid PCR-based method to quantify triplex formation. We have first compared triplex formation on genes located at different genomic sites and containing the same oligopyrimidine*oligopurine sequence. We have shown that efficient TFO binding is possible on both types of genes, expressed and silent. Then we have further investigated when gene transcription may influence triplex formation in chromatin. We have identified situations where for a given gene, increase of transcriptional activity leads to enhanced TFO binding: this was observed for silent or weakly expressed genes that are not or are only slightly accessible to TFO. Such a transcriptional dependence was observed for integrated and endogenous loci, and chemical and biological activations of transcription. Finally, we provide evidence that TFO binding is sequence-specific as measured on mutated target sequences and that up to 50% of chromosomal targets can be covered by the TFO/LNA in living cells.

Figure 1

(A) Sequences of the oligopyrimidine•oligopurine DNA targets and of the oligonucleotides used in this study. Target sequences are indicated in boldface (PPT and VE-CAD), as well as the mutated sequences used as controls (mut_CPPT and mut_GPPT). The 5′-TpA site suitable for psoralen photoadduct formation is shown. Sequences of the psoralen-modified TFO directed against the wild-type PPT duplex (Pso-PPT) and the VE-Cadherin gene (Pso-CAD) are described; small letters indicate LNA nucleotides; cytosines in italic (C and c) are methylated at position 5. (B) Chemical structure of LNA modification.

Figure 2

Quantification of triplex formation. (A) Quantification of triplex formation by Dra I protection assay. (Upper panel) The 16 bp oligopyrimidine•oligopurine target sequence (PPT) present upstream the luciferase gene overlaps a Dra I recognition sequence (boxed); the two arrows indicate the sites of Dra I cleavage. Dra I sites around the PPT sequence are shown and the lengths of the fragments in base pairs obtained after Dra I cleavage are indicated. Location of the RNA probe used for Southern blot analysis is shown. (Lower panel) Naked genomic DNA of CMV(+PPT)/HeLa cells was treated with Pso-PPT and irradiated (+hν) or not (−hν); then the DNA was analyzed by Dra I protection assay (Supplementary Data). DNA markers: (lane M), 1 kb DNA marker (New England Biolabs); (lane P), pCMV(+PPT)/luc plasmid that was used to generate the CMV(+PPT)/HeLa cells, was treated by Pso-PPT, irradiated and cleaved by Dra I. (B) Quantitative PCR analysis of site-directed inter-strand lesions. (Upper panel) Schematic representation of the quantitative PCR analysis method. Regions containing the oligopyrimidine•oligopurine target sequence (Py•Pu) were amplified by quantitative PCR with primers flanking the Py•Pu sequence (primers Tf and Tb). The amounts of these amplified products (‘target fragments’) were compared to that of the amplified products obtained in the same sample from a control region (‘control fragments’) (primers Cf and Cb). (Lower panel) The genomic DNA that was 100% modified by Pso-PPT adducts at the PPT site in the luciferase locus (sample corresponding to lane +hν in Figure 2A) was mixed in various proportions with untreated genomic DNA of CMV(+PPT)/HeLa cells (1:0; 1:0.25; 1:0.5; 1:0.75; 0:1) in order to obtain defined amounts of triplex-induced adducts (with 100, 75, 50, 25, 0%). Relative PCR inhibition (amount of amplified luc target fragments/amount of amplified control fragments) is reported as a function of the amount of triplex-induced adducts in the sample (measured by Dra I protection assay).

Figure 3

Efficient TFO binding is not correlated to gene expression at the target site. Triplex formation was evaluated on PPT sequences present at different genomic loci. The target regions that were PCR-amplified are: luc (integrated luciferase locus), TRβ and CASK genes that all contain the PPT₁₅ sequence, GFP (integrated GFP locus) and COL4A1 genes that contain a mutated PPT sequence (mut_CPPT and mut_GPPT, respectively) (see precise genomic locations in Supplementary Data). (A) Genomic DNA of CMV(+PPT)/HeLa cells was treated with Pso-PPT or Pso-CAD TFO, with or without irradiation and the amount of cross-linked targets was evaluated. (B) CMV(+PPT)/HeLa cells were treated and permeabilized in the presence of Pso-PPT (10 μM) and irradiated. The percentages of triplex-induced adducts on the different genomic targets were reported. Equivalent levels of triplex formation were obtained in nuclei preparations (digitonin-permeabilized cells; closed columns) and in whole cells when Pso-PPT was delivered by SLO permeabilization (hatched columns). The expression status of the target genes was indicated: (+) expressed gene; (−) no detectable expression.

Figure 4

Transcriptional activity influences efficiency of TFO binding in some situations. (A) Triplex formation on the VE-Cadherin gene in different cellular contexts. Percentages of triplex-induced adducts in cell nuclei after treatment with the Pso-CAD (5 μM). Two cells lines were used, either endothelial (EA.hy926) or CMV(+PPT)/HeLa cells, in absence (−) or in presence of TSA treatment (+TSA). VE-cadherin expression at the time of TFO administration was indicated: (+) expressed gene; (−) no detectable expression. (B) Triplex formation on the integrated target in different transcriptional contexts. Percentages of triplex-induced adducts on the PPT target (luciferase gene) and on the mut_GPPT sequence (GFP gene) in cells (closed columns, cell nuclei; hachted columns, whole cells). Before TFO addition, different treatments were performed, as indicated. Relative luciferase RNA levels determined just before Pso-PPT addition were reported; (−) means that the gene is not expressed, with an RT–PCR signal close to the background level.