Stability of DNA Triplexes on Shuttle Vector Plasmids in the Replication Pool in Mammalian Cells

Abstract

Triple helix-forming oligonucleotides may be useful as gene-targeting reagents in vivo, for applications such as gene knockout. One important property of these complexes is their often remarkable stability, as demonstrated in solution and in cells following transfection. Although encouraging, these measurements do not necessarily report triplex stability in cellular compartments that support DNA functions such as replication and mutagenesis. We have devised a shuttle vector plasmid assay that reports the stability of triplexes on DNA that undergoes replication and mutagenesis. The assay is based on plasmids with novel variant supF tRNA genes containing embedded sequences for triplex formation and psoralen cross-linking. Triple helix-forming oligonucleotides were linked to psoralen and used to form triplexes on the plasmids. At various times after introduction into cells, the psoralen was activated by exposure to long wave ultraviolet light (UVA). After time for replication and mutagenesis, progeny plasmids were recovered and the frequency of plasmids with mutations in the supF gene determined. Site-specific mutagenesis by psoralen cross-links was dependent on precise placement of the psoralen by the triple helix-forming oligonucleotide at the time of UVA treatment. The results indicated that both pyrimidine and purine motif triplexes were much less stable on replicated DNA than on DNA in vitro or in total transfected DNA. Incubation of cells with amidoanthraquinone-based triplex stabilizing compounds enhanced the stability of the pyrimidine triplex.Triple helix-forming oligonucleotides (TFOs), 1 or third strands, have received considerable attention because of their potential for intracellular gene targeting (1-11). Many of the biochemical and biophysical properties of triplexes appear to support this application. Triplex formation following the encounter between an appropriate third strand and duplex target has long been recognized as a fundamental structural option of nucleic acids and is not dependent on enzymes or proteins (12). The most stable triplexes are formed on polypurine:polypyrimidine sequences. However, there is considerable stringency with respect to the specific sequence such that single interruptions in a polypurine run can be destabilizing (13-15). Depending on the actual sequence of the purine:pyrimidine run, triplexes can be formed by third strands composed of either purines or pyrimidines (16), which offers some flexibility in TFO design.Once formed, triplexes can be quite stable under appropriate conditions. This was demonstrated some time ago with a pyrimidine triplex with a dissociation half-life of many hours (17). Some purine TFOs form even more stable complexes, with melting temperatures equivalent to or greater than the target duplex (18), and with half-lives of days under optimal conditions (19,20). The persistence of purine motif triplexes formed on DNA fragments and then introduced into mammalian cells has also been measured. A footprinting assay to measure triplexes on transfected...

Targeted Gene Knockout by 2′-O-Aminoethyl Modified Triplex Forming Oligonucleotides

“…1B) (see Levy et al (37), for the construction strategy). Psoralen cross-links placed by the TFO in these positions cause mutations during repair and replication of the plasmid in mammalian cells (17,36). The cross-link site was also positioned in a unique XbaI site that allowed the presence or absence of a cross-link to be determined by restriction enzyme protection.…”

“…Triplex Stability in Vivo-We have described an approach to the measurement of triplex stability in vivo based on a shuttle vector mutation assay (36). The assay reports the presence of a triplex in the nuclear compartment that supports replication and mutagenesis of DNA carrying psoralen cross-links.…”

“…37). In vivo stability assays were performed as described previously (36). Briefly, the plasmid was incubated with individual Pso-TFOs under conditions that supported triplex formation (20 mM Hepes, pH 7.2, 10 mM MgCl 2 , 2 M TFO, 0.6 pmol of plasmid), unbound TFO removed, and the plasmid-TFO complexes were electroporated into Cos-1 cells.…”

Cell Cycle Modulation of Gene Targeting by a Triple Helix-forming Oligonucleotide

“…A second possibility is that the stability of triplexes once formed is much greater in S phase cells than G 0 /G 1 cells. We have shown that triplexes are less stable in cells than in "physiological buffer" in vitro, perhaps because of cellular enzymes that can disrupt triplexes (56). These include helicases and the translocases found in chromatin-remodeling complexes (57)(58)(59).…”

The potential for gene repair via triple helix formation

“…Thermal melting experiments showed that the AE triplexes were more stable than the 2′-Omethyl only triplexes, and, notably, were more stable than the underlying duplex at physiological pH. They were also more stable in the cellular compartment in which replication and mutagenesis occur (62,74). Most importantly they are quite active in the HPRT knockout assay (62).…”