We have targeted the d(GAG)d(CTC) duplex for triplex formation with d(GTG) in the presence of MgCl. The resulting triple helix, d(GTG)*d(GAG)d(CTC), is considerably weaker than the related triplex, d(GAG)*d(GAG)d(CTC), and melts in a biphasic manner, with the third strand dissociating at temperatures about 20-30 °C below that of the remaining duplex. This is in distinct contrast to the d(GAG)*d(GAG)d(CTC) triplex, which melts in essentially a single transition. Gel electrophoresis under non-denaturing conditions shows the presence of the d(GTG)*d(GAG)d(CTC) triplex as a band of low mobility compared to the duplex or the single strand bands. Binding of the d(GTG) third strand and the purine strand of the duplex can be monitored by imino proton NMR spectra. While these spectra are typically very broad for intermolecular triplexes, the line widths can be dramatically narrowed by the addition of two thymines to both ends of the pyrimidine strand. Thermodynamic analysis of UV melting curves shows that this triplex is considerably less stable than related triplexes formed with the same duplex. The orientation of the third strand was addressed by a combination of fluorescence energy transfer and UV melting experiments. Results from these experiments suggest that, in the unlabeled triplex, the preferred orientation of the third strand is parallel to the purine strand of the duplex.

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