Figure 3

Demonstration of specific triplex DNA formation with purine motif oligonucleotides (T1-ap and T2-ap) at the E-1 and E-2 tandem Ets transcription factor binding sites of the human tie-1 promoter by plasmid DNA binding assay. Plasmids containing the tie-1 promoter (ptie-1luc) (see Figure 2B) or 900 bp of the human tie-2 promoter (ptie-2luc) (37) were digested with Smal alone or in combination with AccI (see Figure 1) and incubated overnight with radiolabeled oligonucleotides (T1-ap or T2-ap) at 37 °C in the presence of 10 mM Mg²⁺. Samples were separated on 1% agarose gels containing ethidium bromide (EtBr) and the DNA visualized under UV light (EtBr gel). The gels were dried and autoradiographed to detect triplex DNA formation. (A) Triplex DNA formation of the T1-ap oligonucleotide with intact ptie-1luc plasmid and Smal or AccI ptie-1luc restriction fragments containing the E-1 site. No triplex DNA is observed with T1-ap against ptie-2luc SmaI restriction fragments or with the parallel control oligonucleotide T1-P. (B) Selective binding of T1-ap only to the 770-bp SmaI ptie-1luc restriction fragment containing wildtype E-1 sequence, but not the E-1 mutants (mut-1 and mut-2, see Figure 2B). (C) T2-ap binding to wild-type E-2 present in the same series of wild-type and E-1 mutant (mut-1 and mut-2) SmaI-digested ptie-1luc plasmid fragments. Effect of Mg²⁺ (D) and K⁺ (E) ion concentration on triplex formation with radiolabeled T1-ap and T2-ap.