Exclusion of RNA strands from a purine motif triple helix.

Research concerning oligonucleotide-directed triple helix formation has mainly focused on the binding of DNA oligonucleotides to duplex DNA. The participation of RNA strands in triple helices is also of interest. For the pyrimidine motif (pyrimidine.purine.pyrimidine triplets), systematic substitution of RNA for DNA in one, two, or all three triplex strands has previously been reported. For the purine motif (purine.purine.pyrimidine triplets), studies have shown only that RNA cannot bind to duplex DNA. To extend this result, we created a DNA triple helix in the purine motif and systematically replaced one, two, or all three strands with RNA. In dramatic contrast to the general accommodation of RNA strands in the pyrimidine triple helix motif, a stable triplex forms in the purine motif only when all three of the substituent strands are DNA. The lack of triplex formation among any of the other seven possible strand combinations involving RNA suggests that: (i) duplex structures containing RNA cannot be targeted by DNA oligonucleotides in the purine motif; (ii) RNA strands cannot be employed to recognize duplex DNA in the purine motif; and (iii) RNA tertiary structures are likely to contain only isolated base triplets in the purine motif.     

Mechanism of intermolecular purine-purine-pyrimidine triple helix stabilization by comb-type polylysine graft copolymer at physiologic potassium concentration

We previously reported a novel strategy to stabilize purine motif triplex DNA within a mammalian gene promoter at physiologically relevant pH, temperature, and potassium (K(+)) concentrations by a comb-type poly(L-lysine)-graft-dextran copolymer [Ferdous et al., (1998) Nucleic Acids Res. 26, 3949-3954]. Here we describe the major contribution(s) of the copolymer to stabilize the purine motif triplex DNA at physiological K(+) concentrations. Self-aggregation through guanine-quartet formation of guanine-rich (G-rich) triplex-forming oligonucleotides (TFOs) has long been proposed for K(+)-mediated inhibition of the purine motif triplex formation. However, this was not the case for the severe inhibitory effect of K(+) observed under our reaction conditions. Rather significant decrease in rate of triplex formation involving a G-rich TFO was a major factor to confer K(+) inhibition. Interestingly, in the presence of the copolymer the rate of triplex formation was tremendously increased and K(+)-induced dissociation of preformed triplexes was not observed. Moreover, the triplex-promoting/stabilizing efficiency of the copolymer was amazingly higher than that of physiological concentrations of spermine. An absolute increase in binding constant of the TFO to the target duplex could therefore be the predominant mechanistic source for the copolymer-mediated triplex stabilization under physiological conditions in vitro.     

Poly(L-lysine)-graft-dextran copolymer: amazing effects on triplex stabilization under physiological pH and ionic conditions (in vitro).

Triplex DNA formation involving unmodified triplex-forming oligonucleotides (TFOs) is very unstable under physiological conditions. Here, we report a novel strategy to stabilize both purine and pyrimidine motif triplex DNA within the rat alpha1 (I) collagen gene promoter under physiologically relevant conditions by a poly(L-lysine)- graft -dextran copolymer. Using an in vitro electrophoretic mobility shift assay, we show that the copolymer almost completely abrogates the inhibitory effects of physiological concentrations of monovalent cations, particularly potassium ion (K+), on purine motif triplex formation involving very low concentrations of an unmodified guanine-rich TFO. Of importance, pH dependency in pyrimidine motif triplex formation involving an unmodified cytosine-rich TFO is also significantly overcome by the copolymer. Finally, the triplex-stabilizing efficiency of the copolymer is remarkably higher than that of other oligocations, like spermine and spermidine. We suggest that the ability of the graft copolymer to stabilize triplex DNA under physiologically relevant pH and salt concentrations will be a cue for further progress in the antigene strategy.     

Energetics of strand-displacement reactions in triple helices: a spectroscopic study.

DNA triple helices offer exciting new perspectives toward oligonucleotide-directed inhibition of gene expression. Purine and GT triplexes appear to be the most promising motifs for stable binding under physiological conditions compared to the pyrimidine motif, which forms at relatively low pH. There are, however, very little data available for comparison of the relative stabilities of the different classes of triplexes under identical conditions. We, therefore, designed a model system which allowed us to set up a competition between the oligonucleotides of the purine and pyrimidine motifs targeting the same Watson-Crick duplex. Several conclusions may be drawn: (i) a weak hypochromism at 260 nm is associated with purine triplex formation; (ii) delta H degree of GA, GT and TC triplex formation (at pH 7.0) was calculated as -0.1, -2.5 and -6.1 kcal/mol per base triplet, respectively. This unexpectedly low delta H degree for the purine triple helix formation implies that its delta G degree is nearly temperature-independent and it explains why these triplexes may still be observed at high temperatures. In contrast, the pyrimidine triplex is strongly favoured at lower temperatures; (iii) as a consequence, in a system where two third-strands compete for triplex formation, displacement of the GA or GT strand by a pyrimidine strand may be observed at neutral pH upon lowering the temperature. This original purine-to-pyrimidine triplex conversion shows a significant hypochromism at 260 nm and a hyperchromism at 295 nm which is similar to the duplex-to-triplex conversion in the pyrimidine motif. Further evidence for this triplex-to-triplex conversion is provided by mung bean-nuclease foot-printing assay.     

Targeted correction of an episomal gene in mammalian cells by a short DNA fragment tethered to a triplex-forming oligonucleotide

Triplex-forming oligonucleotides (TFOs) can bind to polypurine/polypyrimidine regions in DNA in a sequence-specific manner and provoke DNA repair. We have coupled a TFO to a short donor fragment of DNA that shares homology to a selected gene as a strategy to mediate gene targeting and correction. In this bifunctional oligonucleotide, the TFO domain is designed to bind the target gene and stimulate repair and recombination, with the donor domain positioned for recombination and information transfer. A series of these tethered donor-TFO (TD-TFO) molecules with donor domains of 40-44 nucleotides and TFO domains in both the purine and pyrimidine triplex motifs were tested for their ability to mediate either gene correction or mutation of a supF reporter gene contained in a SV40 shuttle vector in mammalian cells. In vitro binding assays revealed that the attachment of the donor domain via a flexible linker did not significantly alter the binding affinity of the TFO domain for the polypurine site in the supF target DNA, with equilibrium dissociation constants in the 10(-8) M range. Experiments in which the target vector and the linked TD-TFOs were pre-incubated in vitro and co-transfected into cells led to conversion frequencies approaching 1%, 4-fold greater than with the two domains unlinked. When cells that had been previously transfected with the SV40 vector were electroporated with the TD-TFOs, frequencies of base pair-specific gene correction were seen in the range of 0.04%, up to 50-fold over background and at least 3-fold over either domain alone or in unlinked combinations. Sequence conversion by the TD-TFOs was achieved using either single- or double-stranded donor domains and either triplex motif. Substitution of either domain in the TD-TFO with control sequences yielded reagents with diminished activity, as did mixtures of unlinked TFO and donor DNA segments. The boost in activity provided by the attached TFO domain was reduced in cells deficient in the nucleotide excision repair factor XPA but was restored in a subclone of these cells expressing XPA cDNA, suggesting a role for nucleotide excision repair in the pathway of triple helix-stimulated gene conversion. The ability to correct or mutate a specific target site in mammalian cells using the TD-TFO strategy may provide a useful tool for research and possibly for therapeutic applications.     

Stability of DNA triplexes on shuttle vector plasmids in the replication pool in mammalian cells

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.