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

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 microM ligand (N-[2-(dimethylamino)ethyl]-2-(naphthyl)quinolin-4-ylamine), the binding of CTCTTTTTGCTT (12G) to the sequence GAGAAAAATGAA (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 CTCTTTTTTCTT (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 GAGAAAAAAGAA, 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.

Triple-Helix Formation at Different Positions on Nucleosomal DNA

“…It can be seen that the oligonucleotide still produces a footprint at this site when the DNA is complexed to the nucleosome cores. Parallel triplex formation at this site therefore appears not to be hindered by wrapping the DNA fragment around the nucleosome cores, and contrasts with our previous results with tyrT (43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54) (37). We can exclude the possibility that this footprint (and others described below) is caused by interaction of the oligonucleotide with a small amount of contaminating free DNA since we have shown that in every instance at least 95% of the DNA is associated with the nucleosome cores, even after adding the triplex-forming oligonucleotide.…”

Psoralen Photo-Cross-Linking by Triplex-Forming Oligonucleotides at Multiple Sites in the Human Rhodopsin Gene

“…Although the nucleotides in the linker region of the TFO were designed according to standard antiparallel triplex rules, they were not expected to bind tightly to the underlying duplex because the TFO strand contained many pyrimidine bases, which interact only very weakly, if at all, with the purine-rich strand of the duplex. To assess whether triplex formation occurred in the linker region, we used DNase I footprinting since regions of a duplex that form triplexes are protected from DNase I cleavage ( , ). If nucleotides in the linker were participating in a triplex, the footprint would extend beyond the core footprint produced by a TFO with no additional nucleotides (Figure A, lane 3, TFO3AA).…”

Nucleosome core particles inhibit DNA triple helix formation

“…In this paper we examine the formation of both parallel and antiparallel triplexes at target sites within two fragments derived from tyrT DNA, when reconstituted with nucleosome core particles. Previous studies have demonstrated the formation of parallel triplexes at this target site [26], while the rotational position of the native fragment has been well documented [13].…”

“…The native tyrT DNA fragment contains a run of purines between positions 43 and 54, interrupted by a single thymine at position 46. We changed this residue to adenine by site-directed mutagenesis using the PCR [26], generating tyrT(46A), whose sequence is shown in Figure 1(A). This produces a fragment containing 12 consecutive purine residues, which can be used as a target site for triplex formation using the oligonucleotides shown in Figure 1(B).…”