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

被引:22
作者
Brunet, Erika
Corgnali, Maddalena
Cannata, Fabio
Perrouault, Loic
Giovannangeli, Carine [1 ]
机构
[1] CNRS, UMR 5153, F-75005 Paris, France
[2] INSERM, U565, F-75005 Paris, France
[3] Natl Museum Nat Hist, USM503, F-75005 Paris, France
[4] Univ Udine, Dipartimento Sci & Tecnol Biomed, I-33100 Udine, Italy
关键词
HELIX FORMATION; CROSS-LINKING; LIVING CELLS; GENE; TRANSCRIPTION; CHROMATIN; HETEROCHROMATIN; MODULATION;
D O I
10.1093/nar/gkl630
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 center dot 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.
引用
收藏
页码:4546 / 4553
页数:8
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