Department of Chemistry and Biochemistry, Kent State University , Kent, Ohio 44242, United States.
J Am Chem Soc. 2014 Dec 31;136(52):18062-9. doi: 10.1021/ja510079u. Epub 2014 Dec 15.
The 3' human telomeric overhang provides ample opportunities for the formation and interaction of G-quadruplexes, which have shown impacts on many biological functions including telomerase activities in the telomere region. However, in the few investigations on DNA constructs that approach to the full length of the human telomeric overhang, the presence of higher-order quadruplex-quadruplex interactions is still a subject of debate. Herein, we employed dynamic splint ligation (DSL) to prepare a DNA construct, 5'-(TTAGGG)24 or 24G, which has the length comparable to the full stretch of 3' human telomeric overhang. Using mechanical unfolding assays in laser tweezers, we observed a minor population (∼5%) of higher-order interactions between G-quadruplexes, while the majority of the quadruplexes follow the bead-on-a-string model. Analyses on the noninteracting G-quadruplexes in the 24G construct showed features similar to those of the stand-alone G-quadruplexes in the 5'-(TTAGGG)4 (4G) construct. As each 24G construct contains as many as six G-quadruplexes, this method offers increased throughput for the time-consuming mechanical unfolding experiments of non-B DNA structures.
3' 端人类端粒突出序列为 G-四链体的形成和相互作用提供了充足的机会，这些相互作用对许多生物学功能都有影响，包括端粒区域的端粒酶活性。然而，在少数接近人类端粒突出全长的 DNA 构建体的研究中，高阶四链体-四链体相互作用的存在仍然存在争议。在此，我们采用动态夹板连接（DSL）技术制备了一个 DNA 构建体，其长度与 3' 端人类端粒突出序列的全长相当，即 5'-(TTAGGG)24 或 24G。通过激光镊子的机械解链实验，我们观察到高阶相互作用（约 5%）的小部分群体，而大多数四链体遵循珠串模型。对 24G 构建体中不相互作用的 G-四链体的分析表明，其特征与 5'-(TTAGGG)4（4G）构建体中独立的 G-四链体相似。由于每个 24G 构建体包含多达六个 G-四链体，因此该方法为耗时的非 B-DNA 结构的机械解链实验提供了更高的通量。
