Fossella J A, Kim Y J, Shih H, Richards E G, Fresco J R
Department of Molecular Biology, Princeton University, NJ 08544.
Nucleic Acids Res. 1993 Sep 25;21(19):4511-5. doi: 10.1093/nar/21.19.4511.
The specificity of binding of Watson-Crick base pairs by third strand nucleic acid residues via triple helix formation was investigated in a DNA pyrimidine triplex motif by thermal melting experiments. The host duplex was of the type A10-X-A10: T10-Y-T10, and the third strand T10-Z-T10, giving rise to 16 possible triplexes with Z:XY inserts, 4 duplexes with the Watson-Crick base pairs (XY) and 12 duplexes with mismatch pairs (XZ), all of whose stabilities were compared. Two Z:XY combinations confirm the primary binding of AT and GC target pairs in homopurine.homopyrimidine sequences by T and C residues, respectively. All other Z:XY combinations in the T:AT environment result in triplex destabilization. While some related observations have been reported, the present experiments differ importantly in that they were performed in a T:AT nearest neighbor environment and at physiological ionic strength and pH, all of which were previously untested. The conclusions now drawn also differ substantially from those in previous studies. Thus, by evaluating the depression in Tm due to base triplet mismatches strictly in terms of third strand residue affinity and specificity for the target base pair, it is shown that none of the triplet combinations that destabilize qualify for inclusion in the third strand binding code for the pyrimidine triplex motif. Hence, none of the mismatch triplets afford a general way of circumventing the requirement for homopurine.homopyrimidine targets when third strands are predominated by pyrimidines, as others have suggested. At the same time, the applicability of third strand binding is emphasized by the finding that triplexes are equally or much more sensitive to base triplet mismatches than are Watson-Crick duplexes to base pair mismatches.
通过热变性实验，在DNA嘧啶三链体基序中研究了第三链核酸残基通过三链螺旋形成对沃森-克里克碱基对的结合特异性。宿主双链体为A10-X-A10:T10-Y-T10类型，第三链为T10-Z-T10，产生了16种可能的带有Z:XY插入的三链体、4种带有沃森-克里克碱基对（XY）的双链体和12种带有错配碱基对（XZ）的双链体，比较了它们所有的稳定性。两种Z:XY组合分别证实了在同型嘌呤-同型嘧啶序列中，T和C残基对AT和GC靶碱基对的主要结合。在T:AT环境中的所有其他Z:XY组合都会导致三链体不稳定。虽然已经报道了一些相关观察结果，但本实验的重要不同之处在于，它们是在T:AT最近邻环境以及生理离子强度和pH条件下进行的，而这些条件以前都未经过测试。现在得出的结论也与以前的研究有很大不同。因此，通过严格根据第三链残基对靶碱基对的亲和力和特异性来评估由于碱基三联体错配导致的熔解温度降低，结果表明，任何使三链体不稳定的三联体组合都不符合嘧啶三链体基序的第三链结合密码。因此，正如其他人所建议的，当第三链以嘧啶为主时，没有任何错配三联体能够提供一种普遍的方法来规避对同型嘌呤-同型嘧啶靶标的要求。同时，三链体对碱基三联体错配比沃森-克里克双链体对碱基对错配同样敏感或更敏感这一发现强调了第三链结合的适用性。
