Triplex-induced recombination and repair in the pyrimidine motif

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.

Potential in vivo roles of nucleic acid triple-helices

“…127 TFO-directed mutations were introduced in somatic cells of mice, validating triplex formation in vivo as well as its mutagenic character in several tissues. 128 It appears that the TFO-induced helical alteration of the duplex provokes the mutagenesis 129 and possibly contributes to common chromosomal translocation in cancer (reviewed in ref. 79).…”

Drosophila melanogaster kl-3 and kl-5 Y-loops harbor triple-stranded nucleic acids

“…It has been demonstrated that, upon formation, these structures can frequently downregulate (Cooney et al, 1988; Birg et al, 1990; Faria et al, 2000;Faria et al, 2001) and sometimes upregulate (reviewed in Faria and Giovannangeli, 2001) gene expression, a fact which demonstrates their potential in gene control and suggests their use in gene therapy (Wang et al, 1995;Vasquez et al, 2000) (for a review, see Rogers et al, 2005). The structures are also able to impair DNA polymerization (Dayn et al, 1992), and can influence DNA recombination and repair (Faruqi et al, 1996;Wang et al, 1996;Faruqi et al, 2000;Vasquez et al, 2002;Kalish et al, 2005;Raghavan et al, 2005) (for reviews, see Seidman and Glazer, 2003; Chin et al, 2007). Triplexes might also have a role in chromatin organization of both interphase nuclei (Agazie et al, 1996;Ohno et al, 2002) and mitotic chromosomes .…”

The Lack of Mutagenic Potential of a Guanine-Rich Triplex Forming Oligonucleotide in Physiological Conditions

“…In those studies, the TFOs were designed to specifically bind as a third strand in the antiparallel orientation to the polypurine/polypyrimidine triplex target site (30 bp region) in the supFG1 reporter gene. Although various chemical modifications to the backbone, sugars or bases have been developed to improve the bioactivity of TFOs, we used chemical modifications representative of oligonucleotide-based therapeutics currently being investigated in the clinic (Kalish et al, 2005).…”