Alternate-strand DNA triple-helix formation using short acridine-linked oligonucleotides.
Alternate-strand DNA triple-helix formation using short acridine-linked oligonucleotides.
复制标题
使用短吖啶连接的寡核苷酸形成交替链 DNA 三螺旋。
作者：
E. Washbrook;K. Fox
我们已经使用DNAse I足迹来检查分子间DNA三螺旋在含有相邻嘌呤和嘧啶块的序列上的形成。目标位点G6T6。A6C6和T6G6。将C6A6克隆成较长的DNA片段，作为DNAse I足迹的底物，分别检测了Acr-连接的寡核苷酸Acr- t5g5和Acr- g5t5的结合。这第三条链被设计成包括G.GC三联链和T.AT三联链，前者是由反向胡格斯汀碱基对连接在一起的反平行的Gn链，后者是两条含有t的链彼此反平行排列的。我们发现Acr-T5G5与目标序列G6T6结合。-A6C6，在pH 7.0的镁存在下，产生清晰的dna酶I足迹。在这个结构中，中央鸟嘌呤不被第三链识别，可以被硫酸二甲酯修饰。在这些条件下，Acr-G5T5和T6G6没有观察到足迹。C6A6，虽然在氯化锰的存在下这个三联体是很明显的。锰还促进了Acr-T5G5与含有序列T6G6.C6A6的片段中的第二个位点的结合。这代表了与序列G4ATCT6的相互作用，G4ATCT6位于合成插入物和片段剩余部分之间的边界，并表明这种二价金属离子可能稳定含有一个或两个不匹配的三联体。锰不影响两种寡核苷酸与G6T6.A6C6的相互作用。
We have used DNAse I footprinting to examine the formation of intermolecular DNA triple helices at sequences containing adjacent blocks of purines and pyrimidines. The target sites G6T6.A6C6 and T6G6.C6A6 were cloned into longer DNA fragments and used as substrates for DNAse I footprinting, which examined the binding of the acridine (Acr)-linked oligonucleotides Acr-T5G5 and Acr-G5T5 respectively. These third strands were designed to incorporate both G.GC triplets, with antiparallel Gn strands held together by reverse Hoogsteen base pairs, and T.AT triplets, with the two T-containing strands arranged antiparallel to each other. We find that Acr-T5G5 binds to the target sequence G6T6.-A6C6, in the presence of magnesium at pH 7.0, generating clear DNAse I footprints. In this structure the central guanine is not recognized by the third strand and is accessible to modification by dimethyl sulphate. Under these conditions no footprint was observed with Acr-G5T5 and T6G6.C6A6, though this triplex was evident in the presence of manganese chloride. Manganese also facilitated the binding of Acr-T5G5 to a second site in the fragment containing the sequence T6G6.C6A6. This represents interaction with the sequence G4ATCT6, located at the boundary between the synthetic insert and the remainder of the fragment, and suggests that this bivalent metal ion may stabilize triplexes that contain one or two mismatches. Manganese did not affect the interaction of either oligonucleotide with G6T6.A6C6.