Zhang Xiaonong, Zhang Yingqi, Zhang Wenke
State Key Laboratory of Supramolecular Structure and Materials, Jilin University, Changchun 130012, People's Republic of China.
Nucleic Acids Res. 2020 Jul 9;48(12):6458-6470. doi: 10.1093/nar/gkaa479.
The dynamic topological structure of telomeric DNA is closely related to its biological function; however, no such structural information on full-length telomeric DNA has been reported due to difficulties synthesizing long double-stranded telomeric DNA. Herein, we developed an EM-PCR and TA cloning-based approach to synthesize long-chain double-stranded tandem repeats of telomeric DNA. Using mechanical manipulation assays based on single-molecule atomic force microscopy, we found that mechanical force can trigger the melting of double-stranded telomeric DNA and the formation of higher-order structures (G-quadruplexes or i-motifs). Our results show that only when both the G-strand and C-strand of double-stranded telomeric DNA form higher-order structures (G-quadruplexes or i-motifs) at the same time (e.g. in the presence of 100 mM KCl under pH 4.7), that the higher-order structure(s) can remain after the external force is removed. The presence of monovalent K+, single-wall carbon nanotubes (SWCNTs), acidic conditions, or short G-rich fragments (∼30 nt) can shift the transition from dsDNA to higher-order structures. Our results provide a new way to regulate the topology of telomeric DNA.
端粒DNA的动态拓扑结构与其生物学功能密切相关；然而，由于合成长双链端粒DNA存在困难，尚未有关于全长端粒DNA的此类结构信息的报道。在此，我们开发了一种基于EM-PCR和TA克隆的方法来合成端粒DNA的长链双链串联重复序列。使用基于单分子原子力显微镜的机械操纵试验，我们发现机械力可以触发双链端粒DNA的解链以及高阶结构（G-四链体或i-基序）的形成。我们的结果表明，只有当双链端粒DNA的G链和C链同时形成高阶结构（G-四链体或i-基序）时（例如在pH 4.7、100 mM KCl存在的情况下），外力去除后高阶结构才能保留。单价K+、单壁碳纳米管（SWCNT）、酸性条件或短的富含G的片段（约30 nt）的存在可以改变从双链DNA到高阶结构的转变。我们的结果为调节端粒DNA的拓扑结构提供了一种新方法。
