Nucleic Acids Research Advance Access published online on September 1, 2006
Nucleic Acids Research, doi:10.1093/nar/gkl630
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© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Molecular Biology |
Targeting chromosomal sites with locked nucleic acid-modified triplex-forming oligonucleotides: study of efficiency dependence on DNA nuclear environment
1 CNRS, UMR5153 Paris, F-75005, France 2 Inserm, U565 Paris, F-75005, France 3 Museum National d'Histoire Naturelle, USM503 Paris, F-75005, France 4 Dipartimento di Scienze e Tecnologie Biomediche, Universita degli Studi di Udine 33100 Udine, Italy
*To whom correspondence should be addressed. Tel: +33 1 40793711; Fax: + 33 1 40793705; Email: giovanna{at}mnhn.fr
Received May 8, 2006. Revised August 7, 2006. Accepted August 10, 2006.
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.
Present address: Erika Brunet, Memorial Sloan Kettering Cancer Center, New York, 10021 NY, USA
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