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We have used DNase I footprinting to examine the effect of a triplex-binding ligand on the formation of parallel intermolecular DNA triple helices at a mixed sequence target site contained within a natural DNA fragment (tyrT). In the presence of 10 microM ligand (N-[2-(dimethylamino)ethyl]-2-(naphthyl)quinolin-4-ylamine), the binding of CTCGCTT (12G) to the sequence GAGTGAA (generating a complex containing 8 x T x AT, 1 x G x TA and 3 x C+ x GC triplets) was enhanced 3-fold at pH 5.5. When the oligonucleotide CTCCTT (12T) was substituted for 12G (replacing G x TA with T x TA) there was a large reduction in affinity for the target sequence. However, this was stabilized by about 300-fold in the presence of the ligand, requiring a similar concentration to produce a footprint as 12G in the absence of the ligand. When the sequence of the target site was altered to GAGGAA, generating an uninterrupted run of purines [tyrT(46A)], the binding of 12T (generating a complex containing 9 x T x AT, and 3 x C+ x GC triplets) was enhanced 3-fold by 10 microM of the triplex-binding ligand. However, although the binding of 12G to this sequence generating a complex containing a G x AT triplet, was much weaker, this too was stabilized by about 30-fold by the ligand, requiring a similar concentration as the perfect matched oligonucleotide (12T) in the absence of the ligand. A secondary, less stable footprint was also observed in these fragments when using either 12T or 12G, which was evident only in the presence of the triplex-binding ligand. This site, which contained a number of triplet mismatches, appears to be realated to the formation of four or five central T x AT triplets. This reduction in the stringency of oligonucleotide binding by the triplex-binding ligand promotes the formation of complexes at non-targeted regions but may also have the potential for enabling recognition at sites that contain regions where there are no specific triplet matches.

Effect of a triplex-binding ligand on triple helix formation at a site within a natural DNA fragment