Cherny D I, Malkov V A, Volodin A A, Frank-Kamenetskii M D
Institute of Molecular Genetics, Russian Academy of Sciences, Moscow.
J Mol Biol. 1993 Mar 20;230(2):379-83. doi: 10.1006/jmbi.1993.1154.
Using biotinylated oligonucleotides and streptavidin as a marker, we have visualized, with the help of electron microscopy, the triplex formation. We used the natural homopurine-homopyrimidine sequence from human papillomavirus 16 cloned within a plasmid. Under conditions favouring the formation of pyrimidine-purine-pyrimidine triplex the corresponding pyrimidine oligonucleotide formed a complex with the insert and this complex was detected by electron microscopy. Similarly, under conditions favouring the formation of pyrimidine-purine-purine triplex the corresponding purine oligonucleotide formed a stable complex detected by electron microscopy. In both cases the complexes we observed exhibited remarkable sequence specificity. Near 80% of DNA molecules carried the streptavidin marker in the correct position and very few cases of non-specific binding were detected. We conclude that the triplex mode of recognition may provide very efficient sequence-specific markers for electron microscopy of DNA.
我们使用生物素化的寡核苷酸和链霉亲和素作为标记物，借助电子显微镜观察到了三链体的形成。我们使用了克隆在质粒中的人乳头瘤病毒16的天然同型嘌呤-同型嘧啶序列。在有利于嘧啶-嘌呤-嘧啶三链体形成的条件下，相应的嘧啶寡核苷酸与插入片段形成复合物，并且通过电子显微镜检测到了该复合物。同样，在有利于嘧啶-嘌呤-嘌呤三链体形成的条件下，相应的嘌呤寡核苷酸形成了一种稳定的复合物，该复合物也通过电子显微镜检测到。在这两种情况下，我们观察到的复合物都表现出显著的序列特异性。近80%的DNA分子在正确的位置携带链霉亲和素标记物，并且检测到的非特异性结合情况很少。我们得出结论，三链体识别模式可能为DNA的电子显微镜观察提供非常有效的序列特异性标记物。
