Cassidy S A, Strekowski L, Wilson W D, Fox K R
Department of Physiology & Pharmacology, University of Southampton, U.K.
Biochemistry. 1994 Dec 27;33(51):15338-47. doi: 10.1021/bi00255a015.
We have used DNase I footprinting to investigate the effect of a triplex-binding ligand on the formation of intermolecular DNA triple helices at target sites that have been cloned into longer DNA fragments. In the presence of a triplex-binding ligand (N-[2-(dimethylamino)ethyl]-2-(2-naphthyl)quinolin-4-ylamine ), the concentrations of T5C5 and C5T5 required to generate DNase I footprints at the target sites A6G6.C6T6 and G6A6.T6C6, respectively, are reduced by at least 100-fold. Complexes with the acridine-linked oligonucleotides Acr-T5C5 and Acr-C5T5 are stabilized to a much lesser extent and produce footprints at concentrations similar to those of the unmodified oligonucleotides in the presence of the ligand. The stabilizing effects of acridine modification or the addition of a triplex-binding ligand are not additive. The position and length of the footprints produced by Acr-T5C5 and T5C5 at the target sequence A6G6.C6T6 are unaffected by the ligand. In contrast, footprints at the target site G6A6.T6C6 appear 3-4 bases shorter in the presence of the ligand, when viewed from the pyrimidine strand, and 1-2 bases longer on the purine strand. These results are explained by suggesting that the compound binds at T.AT triplets and prevents the transmission of any DNA structural changes into the flanking duplex. The compound has a smaller stabilizing effect on short antiparallel triplexes consisting of G.GC and T.AT triplets. Binding of Acr-G5T5 to A6G6.C6T6 is enhanced slightly by the compound, which increases the apparent footprinting site, probably by preventing fraying at the 3'-end of the third strand. The compound does not promote the binding of G5T5 to A6G6.C6T6 or that of Acr-T5G5 and T5G5 to G6A6.T6C6.
我们利用DNA酶I足迹法来研究三链结合配体对已克隆到较长DNA片段中的靶位点处分子间DNA三链螺旋形成的影响。在存在三链结合配体（N-[2-(二甲基氨基)乙基]-2-(2-萘基)喹啉-4-胺）的情况下，分别在靶位点A6G6.C6T6和G6A6.T6C6处产生DNA酶I足迹所需的T5C5和C5T5浓度至少降低了100倍。与吖啶连接的寡核苷酸Acr-T5C5和Acr-C5T5形成的复合物稳定程度要小得多，并且在存在配体的情况下，产生足迹的浓度与未修饰寡核苷酸的浓度相似。吖啶修饰或添加三链结合配体的稳定作用并非相加的。Acr-T5C5和T5C5在靶序列A6G6.C6T6处产生的足迹的位置和长度不受配体影响。相比之下，从嘧啶链看，在存在配体的情况下，靶位点G6A6.T6C6处的足迹短3 - 4个碱基，从嘌呤链看则长1 - 2个碱基。这些结果可以解释为该化合物结合在T.AT三联体处，并阻止任何DNA结构变化传递到侧翼双链体中。该化合物对由G.GC和T.AT三联体组成的短反平行三链体的稳定作用较小。该化合物略微增强了Acr-G5T5与A6G6.C6T6的结合，这可能通过防止第三条链3'端的解链来增加表观足迹位点。该化合物不会促进G5T5与A6G6.C6T6的结合，也不会促进Acr-T5G5和T5G5与G6A6.T6C6的结合。
