Figure 1.

(A) Experimental design. The PGK/luc plasmids contain the firefly luciferase gene (luc) under control of the PGK promoter. An insert of 34 bp containing either the wild-type PPT [(+)PPT/luc] or a mutated PPT [(+)PPTmut/luc] has been cloned within the 5′ transcribed but untranslated region of the luciferase gene. Alternatively, there was no insertion of a 34-bp fragment [(−)PPT/luc]. Three sequences of 15-mer np oligonucleotides were used in the described experiments. The TFO (15TCG) perfectly matches the wild-type PPT. Two control oligonucleotides were used (15-I1 and 15-I2). The cytosines were methylated at the C5 position (in italics). Two 5′ substituted conjugates containing an acridine derivative (Acr-15TCG) or a psoralen derivative (Pso-15TCG) were used in some of the experiments. (B) Specificity of triplex formation. The double-stranded target containing the wild-type PPT [(+)PPT] or mutated PPT [(+)PPTmut] (for sequences see Materials and Methods) labeled on the 5′ end of the pyrimidine strand was incubated overnight in a buffer containing 50 mM Hepes, 50 mM NaCl, 10 mM MgCl₂ in the presence of increasing concentrations (as indicated on the top of the gels) of 15TCG or control 15-I1 np oligonucleotide. The samples were analyzed by electrophoresis in a 15% nondenaturing polyacrylamide gel (50 mM Hepes, 10 mM MgCl₂) at 37°C. The left lane on each gel corresponds to the radiolabeled pyrimidine strand alone (Py*). D, duplex; T, triplex.