Journal article
Triplex-induced recombination and repair in the pyrimidine motif
Nucleic acids research, Vol.33(11), pp.3492-3502
2005
DOI: 10.1093/nar/gki659
PMCID: PMC1151591
PMID: 15961731
Abstract
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.
Details
- Title: Subtitle
- Triplex-induced recombination and repair in the pyrimidine motif
- Creators
- Jennifer M KalishMichael M SeidmanDaniel L WeeksPeter M Glazer
- Resource Type
- Journal article
- Publication Details
- Nucleic acids research, Vol.33(11), pp.3492-3502
- Publisher
- Oxford University Press
- DOI
- 10.1093/nar/gki659
- PMID
- 15961731
- PMCID
- PMC1151591
- ISSN
- 0305-1048
- eISSN
- 1362-4962
- Language
- English
- Date published
- 2005
- Academic Unit
- Stead Family Department of Pediatrics; Biochemistry and Molecular Biology
- Record Identifier
- 9984024504902771
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