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

Brown, Philip M., Drabble, Amelia and Fox, Keith R. (1996) Effect of a triplex-binding ligand on triple helix formation at a site within a natural DNA fragment. Biochemical Journal, 314 (2), 427-432. (doi:10.1042/bj3140427).

Abstract

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 μM ligand (N-[2-(dimethylamino)ethyl]-2-(2-naphthyl)quinolin-4-yl-amine), the binding of CTCTTTTTGCTT (12G) to the sequence GAGAAAAATGAA (generating a complex containing 8×T·AT, 1×G·TA and 3×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×T·AT, and 3×C⁺·GC triplets) was enhanced 3-fold by 10 μM of the triplex-binding ligand. However, although the binding of 12G to this sequence, generating a complex containing a G·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 related to the formation of four or five central T·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.

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Contributors

Author: Keith R. Fox

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