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

We have used DNase I footprinting to examine the effect of a tripler-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 mu M ligand (N-[2-(dimethylamino)ethyl]-2-(2-naphthyl)quinolin-4-ylamine), the binding of CTCTTTTTGCTT (12G) to the sequence GAGAAAAATGAA (generating a complex containing 8xT . AT, 1 x G . TA and 3 x C+ . GC triplets) was enhanced 3-fold at pH 5.5. When the oligonucleotide CTCTTTTTTCTT (12T) was substituted for 12G (replacing G . TA with T . 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 GAGAAAAAAGAA, generating an uninterrupted run of purines [tyrT(46A)], the binding of 12T (generating a complex containing 9 x T . AT, and 3 x C+ . GC triplets) was enhanced 3-fold by 10 mu M of the tripler-binding ligand. However, although the binding of 12G to this sequence, generating a complex containing a G . AT tripler, 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 tripler-binding ligand. This site, which contained a number of triplet mismatches, appears to be related to the formation of four or five central T . AT triplets. This reduction in the stringency of oligonucleotide binding by the tripler-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 tripler matches.