The dynamics of forming a triplex in an artificial telomere inferred by DNA mechanics
The dynamics of forming a triplex in an artificial telomere inferred by DNA mechanics
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DNA力学推断的人工端粒形成三链体的动力学
DOI：
10.1093/nar/gkz464
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发表时间：
2019-09-05
影响因子：
14.9
通讯作者：
Yu，Zhongbo
中科院分区：
文献类型：
--
作者：
Li，Ning;Wang，Junli;Yu，Zhongbo
摘要端粒携带重复序列，末端为单链突出端。富含G的突出端可以折叠并结合在其上游双链体的大沟中，形成反平行的三链体结构。端粒三链体已被提出在保护染色体末端中起作用。然而，我们缺乏策略来机械探测端粒三链体的动力学。在这里，我们表明，端粒三链体的拓扑动力学涉及3′突出结合在ds/ssDNA连接推断的DNA力学。在点击化学和分支聚合酶链反应的辅助下，我们开发了一种机械操纵具有自由末端的人工端粒DNA的救援绳策略。使用单分子磁镊，我们确定了一个很少形成（5%）端粒三链体，在解压缩沃森-克里克配对双链体时暂停在中间状态。我们的研究结果表明，在端粒DNA中形成的机械稳定的三链体可以在生理缓冲液中抵抗20 pN的力几秒钟。我们还证明了救援绳策略辅助的机械操作可以直接破坏DNA三链体中第三链与其靶向双链体之间的相互作用。我们的单分子救援绳策略将作为研究端粒动力学和进一步开发基于三链体的生物技术的通用工具。
Abstract A telomere carrying repetitive sequences ends with a single-stranded overhang. The G-rich overhang could fold back and bind in the major groove of its upstream duplex, forming an antiparallel triplex structure. The telomeric triplex has been proposed to function in protecting chromosome ends. However, we lack strategies to mechanically probe the dynamics of a telomeric triplex. Here, we show that the topological dynamics of a telomeric triplex involves 3′ overhang binding at the ds/ssDNA junction inferred by DNA mechanics. Assisted by click chemistry and branched polymerase chain reaction, we developed a rescue-rope-strategy for mechanically manipulating an artificial telomeric DNA with a free end. Using single-molecule magnetic tweezers, we identified a rarely forming (5%) telomeric triplex which pauses at an intermediate state upon unzipping the Watson–Crick paired duplex. Our findings revealed that a mechanically stable triplex formed in a telomeric DNA can resist a force of 20 pN for a few seconds in a physiological buffer. We also demonstrated that the rescue-rope-strategy assisted mechanical manipulation can directly rupture the interactions between the third strand and its targeting duplex in a DNA triplex. Our single-molecule rescue-rope-strategy will serve as a general tool to investigate telomere dynamics and further develop triplex-based biotechnologies.