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In most cases, unmodified oligonucleotides designed as antigene molecules are incapable of binding to DNA with sufficient stability to prevent gene expression. To stabilize binding to a polypurine tract in the HER-2/neu promoter, a triplex forming oligonucleotide (TFO) was conjugated to a pyrrolo[1,4]benzodiazepine (PBD), desmethyltomaymycin, and site-specific DNA binding was evaluated. An activated ester of the PBD moiety was conjugated by an acylation reaction to a free primary amine on a 50-atom aliphatic linker at the 5' end of the TFO. This long aliphatic linker was designed to provide a bridge from the major groove binding site of the TFO to the minor groove binding site of the PBD. Triplex formation by the resulting TFO-PBD conjugate occurred more slowly and with a nearly 30-fold lower affinity compared to an unconjugated TFO. PBD binding to the triplex target was demonstrated by protection from restriction enzyme digestion, and covalent binding to the exocyclic amino group of guanine was inferred by substituting specific guanines with inosines. Although the binding of the TFO was less efficient, this report demonstrates that in principle, TFOs can be used to direct the binding of a PBD to specific location. Further optimization of TFO-PBD conjugate design, likely involving optimization of the linker and perhaps placing a PBD at both ends of the TFO, will be needed to make gene modification robust.

In vitro assembly of an intermolecular purine*purine.pyrimidine triple helix requires the presence of a divalent cation. The relationships between cation coordination and triplex assembly were investigated, and we have obtained new evidence for at least three functionally distinct potential modes of divalent cation coordination. (i) The positive influence of the divalent cation on the affinity of the third strand for its specific target correlates with affinity of the cation for coordination to phosphate. (ii) Once assembled, the integrity of the triple helical structure remains dependent upon its divalent cation component. A mode of heterocyclic coordination/chelation is favorable to triplex formation by decreasing the relative tendency for efflux of integral cations from within the triple helical structure. (iii) There is also a detrimental mode of base coordination through which a divalent cation may actively antagonize triplex assembly, even in the presence of other supportive divalent cations. These results demonstrate the considerable impact of the cationic component, and suggest ways in which the triple helical association might be positively or negatively modulated.

Triplex formation may be of potential utility to inhibit the expression of individual genes. We describe the formation of a triple helix in the coding sequence of the HER-2/neu gene. In vitro transcription analysis in the presence and absence of triplex formation demonstrates that an unmodified DNA triplex-forming oligonucleotide is incapable of inhibiting RNA polymerase elongation. Triplex formation by an oligonucleotide-psoralen conjugate was used to form a covalent photoadduct with a thymine on the nontemplate strand of the HER-2/neu gene. In the native HER-2/neu gene, covalent attachment of the triplex-forming oligonucleotide to the nontemplate strand did not prevent RNA polymerase elongation. Using HER-2/neu point mutants that would place the target thymine on the template strand, we demonstrated that covalent modification of the template strand was necessary to inhibit RNA polymerase elongation. Based on these data, we synthesized oligonucleotide-alkylator conjugates that would react with a specific guanine residue on the template strand of the HER-2/neu coding sequence. The oligonucleotide-alkylator conjugates inhibited transcription elongation by T7 RNA polymerase and eukaryotic RNA polymerase II from a HeLa nuclear extract. These studies demonstrate the successful application of triplex-forming oligonucleotide-alkylator conjugates to inhibit transcription elongation in the HER-2/neu gene, and show that covalent modification of the DNA strand used as the transcription template is necessary to prevent RNA polymerase elongation.

Triplex-forming oligonucleotides (TFOs) have been shown to bind to target DNA sequences in several human gene promoters such as the c-myc oncogene, the epidermal growth factor receptor, and the dihydrofolate reductase genes. TFOs have been shown to inhibit transcription in vitro and gene expression in cell culture of the c-myc and other genes. The HER-2/neu oncogene, which is overexpressed in breast cancer and other human malignancies, contains a purine-rich sequence in its promoter, which is favorable for purine:purine:pyrimidine (R:R:Y) triplex formation. Although its function in the HER-2/neu promoter is unknown, this purine-rich site is homologous to a protein-binding sequence in the promoter of the epidermal growth factor receptor that is necessary for efficient transcription of this gene. We have shown that this sequence is a site for nuclear protein binding by incubation with a crude nuclear extract. We describe the formation of an interstrand triplex using a purine-rich oligonucleotide antiparallel to this purine-rich target sequence of the HER-2/neu promoter. Triplex formation by the oligonucleotide prevents protein binding to the target site in the HER-2/neu promoter in vitro. We have shown that this oligonucleotide is a potent and specific inhibitor of HER-2/neu transcription in an in vitro assay. The triplex target site contains a single pyrimidine base that does not conform to the R:R:Y triplex motif. In an attempt to abrogate the potentially destabilizing effects of this pyrimidine base on triplex formation, we have substituted an abasic linker for the pyrimidine residue in the triplex forming oligonucleotide. Triplex formation with the modified oligonucleotide appears to occur with approximately equivalent binding affinity. Triplex formation in the HER-2/neu oncogene promoter prevents transcription in vitro and may represent a future modality for specific inhibition of this gene in vivo.