Brown P M, Drabble A, Fox K R
Department of Physiology and Pharmacology, University of Southampton, U.K.
Biochem J. 1996 Mar 1;314 ( Pt 2)(Pt 2):427-32. doi: 10.1042/bj3140427.
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.
我们利用脱氧核糖核酸酶I足迹法来研究三链体结合配体对天然DNA片段（tyrT）中混合序列靶位点处平行分子间DNA三链体形成的影响。在存在10微摩尔配体（N-[2-(二甲基氨基)乙基]-2-(萘基)喹啉-4-胺）的情况下，CTCTTTTTGCTT（12G）与序列GAGAAAAATGAA的结合（形成包含8个T×AT、1个G×TA和3个C⁺×GC三联体的复合物）在pH 5.5时增强了3倍。当用寡核苷酸CTCTTTTTTCTT（12T）替代12G（用T×TA取代G×TA）时，对靶序列的亲和力大幅降低。然而，在配体存在下，这种亲和力稳定了约300倍，产生足迹所需的浓度与不存在配体时12G产生足迹所需的浓度相似。当靶位点序列改变为GAGAAAAAAGAA，形成不间断的嘌呤链[tyrT(46A)]时，12T（形成包含9个T×AT和3个C⁺×GC三联体的复合物）的结合在10微摩尔三链体结合配体作用下增强了3倍。然而，尽管12G与该序列结合形成包含一个G×AT三联体的复合物的能力弱得多，但它也因配体而稳定了约30倍，产生足迹所需的浓度与不存在配体时完美匹配的寡核苷酸（12T）相似。当使用12T或12G时，在这些片段中还观察到一个次要的、稳定性较差的足迹，仅在存在三链体结合配体时才明显。这个位点包含一些三联体错配，似乎与四个或五个中心T×AT三联体的形成有关。三链体结合配体降低寡核苷酸结合的严格性，促进了非靶区域复合物的形成，但也可能有潜力使在没有特定三联体匹配区域的位点实现识别。
