Published online 16 June 2005
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Triplex-induced recombination and repair in the pyrimidine motif
1Department of Therapeutic Radiology, Yale University School of Medicine PO Box 208040, HRT 140, New Haven, CT 06520-8040, USA 2Department of Genetics, Yale University School of Medicine PO Box 208040, HRT 140, New Haven, CT 06520-8040, USA 3National Institute on Aging, National Institutes of Health 5600 Nathan Shock Drive, Baltimore, MD 21224, USA 4Department of Biochemistry, University of Iowa Iowa City, IA 52242, USA
*To whom correspondence should be addressed. Tel: +1 203 737 2788; Fax: +1 203 785 6309; Email: peter.glazer{at}yale.edu
Received May 2, 2005. Revised May 27, 2005. Accepted May 27, 2005.
Triplex-forming oligonucleotides (TFOs) bind DNA in a sequence-specific manner at polypurine/polypyrimidine sites and mediate targeted genome modification. Triplexes are formed by either pyrimidine TFOs, which bind parallel to the purine strand of the duplex (pyrimidine, parallel motif), or purine TFOs, which bind in an anti-parallel orientation (purine, anti-parallel motif). Both purine and pyrimidine TFOs, when linked to psoralen, have been shown to direct psoralen adduct formation in cells, leading to mutagenesis or recombination. However, only purine TFOs have been shown to mediate genome modification without the need for a targeted DNA-adduct. In this work, we report the ability of a series of pyrimidine TFOs, with selected chemical modifications, to induce repair and recombination in two distinct episomal targets in mammalian cells in the absence of any DNA-reactive conjugate. We find that TFOs containing N3'
P5' phosphoramidate (amidate), 5-(1-propynyl)-2'-deoxyuridine (pdU), 2'-O-methyl-ribose (2'-O-Me), 2'-O-(2-aminoethyl)-ribose, or 2'-O, 4'-C-methylene bridged or locked nucleic acid (LNA)-modified nucleotides show substantially increased formation of non-covalent triplexes under physiological conditions compared with unmodified DNA TFOs. However, of these modified TFOs, only the amidate and pdU-modified TFOs mediate induced recombination in cells and stimulate repair in cell extracts, at levels comparable to those seen with purine TFOs in similar assays. These results show that amidate and pdU-modified TFOs can be used as reagents to stimulate site-specific gene targeting without the need for conjugation to DNA-reactive molecules. By demonstrating the potential for induced repair and recombination with appropriately modified pyrimidine TFOs, this work expands the options available for triplex-mediated gene targeting.
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