Triplex formation by oligonucleotides containing 5-(1-propynyl)-2'-deoxyuridine: decreased magnesium dependence and improved intracellular gene targeting.

Oligonucleotides capable of sequence-specific triple helix formation have been proposed as DNA binding ligands useful for modulation of gene expression and for directed genome modification. However, the effectiveness of such triplex-forming oligonucleotides (TFOs) depends on their ability to bind to their target sites within cells, and this can be limited under physiologic conditions. In particular, triplex formation in the pyrimidine motif is favored by unphysiologically low pH and high magnesium concentrations. To address these limitations, a series of pyrimidine TFOs were tested for third-strand binding under a variety of conditions. Those containing 5-(1-propynyl)-2'-deoxyuridine (pdU) and 5-methyl-2'-deoxycytidine (5meC) showed superior binding characteristics at neutral pH and at low magnesium concentrations, as determined by gel mobility shift assays and thermal dissociation profiles. Over a range of Mg2+ concentrations, pdU-modified TFOs formed more stable triplexes than did TFOs containing 2'-deoxythymidine. At 1 mM Mg2+, a DeltaTm of 30 degreesC was observed for pdU- versus T-containing 15-mers (of generic sequence 5' TTTTCTTTTTTCTTTTCT 3') binding to the cognate A:T bp rich site, indicating that pdU-containing TFOs are capable of substantial binding even at physiologically low Mg2+ concentrations. In addition, the pdU-containing TFOs were superior in gene targeting experiments in mammalian cells, yielding 4-fold higher mutation frequencies in a shuttle vector-based mutagenesis assay designed to detect mutations induced by third-strand-directed psoralen adducts. These results suggest the utility of the pdU substitution in the pyrimidine motif for triplex-based gene targeting experiments.

Inhibition of transcription of the human c-myc protooncogene by intermolecular triplex.

Triplex-forming oligonucleotides (TFOs) have been shown to inhibit both transcription in vitro and the expression of target genes in cell culture by binding to polypurine/polypyrimidine sequences in several human gene promoters. The c-myc protooncogene is overexpressed in a variety of human cancers and appears to play an important role in the proliferation of these cells. In an attempt to assay the ability of triplex-forming oligonucleotides to inhibit expression of a target gene in vivo, we have developed a cellular system involving transfection of a c-myc promoter-driven luciferase reporter plasmid with triplex-forming oligonucleotides targeted to the human c-myc protooncogene. To increase the stability of the TFO, we have used modified phosphorothioate oligonucleotides. Triplex formation with a modified phosphorothioate oligonucleotide occurs with approximately equal binding affinity as that seen using a phosphodiester oligonucleotide. Phosphorothioate-modified TFOs targeted to c-myc inhibit transcription of the c-myc promoter in HeLa cells as demonstrated by a decrease in luciferase expression from a luciferase reporter gene construct. These results suggests that triplex formation may represent a gene-specific means of inhibiting specific protooncogene expression.

Inhibition of nuclear protein binding to two sites in the murine c-myc promoter by intermolecular triplex formation.

The c-myc gene is overexpressed in a variety of tumor types and appears to play an important role in the abnormal growth of a number of cell types. In an effort to determine the ability of sequence- and species-specific triplex-forming oligonucleotides to inhibit expression of a targeted gene in animals, we have identified two novel triplex-forming sites in the murine c-myc promoter. One is homologous to the triplex-forming human PuF binding element located upstream of the P1 transcription start site. The other triplex-forming site is found in a region between P1 and P2 that encompasses the ME1a1 binding site and part of the E2F binding site and is highly homologous to the human sequence. Synthetic oligodeoxyribonucleotides designed to target these essential regulatory elements form sequence-specific triple helices as demonstrated by gel mobility shift analysis and DNase I footprinting. Polypurine: polypyrimidine regions in the P1 and P2 promoters form specific protein-DNA complexes upon incubation with a murine YC8 nuclear extract. Preincubation of each of the promoter fragments with its respective triplex-forming oligonucleotide results in the inhibition of nuclear protein binding. Non-triplex-forming oligonucleotides do not significantly affect protein binding. The data presented are a preliminary step toward generating an animal model for the phenotypic effects of triplex formation within the c-myc promoter.