Conformations of Human Telomeric G-Quadruplex Studied Using a Nucleotide-Independent Nitroxide Label.
Conformations of Human Telomeric G-Quadruplex Studied Using a Nucleotide-Independent Nitroxide Label.
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DOI：
10.1021/acs.biochem.5b01189
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发表时间：
2016-01-19
期刊：
影响因子：
2.9
通讯作者：
Qin PZ
中科院分区：
文献类型：
--
作者：
Zhang X;Xu CX;Di Felice R;Sponer J;Islam B;Stadlbauer P;Ding Y;Mao L;Mao ZW;Qin PZ
富含鸟嘌呤的寡核苷酸可以形成独特的G-四链体（GQ）结构，其具有通过Hoogsteen键合在平面中组织的四个鸟嘌呤碱基的堆叠单元。已经在体内检测到GQ结构，并显示其在维持基因组完整性和调节基因表达中发挥作用。了解GQ构象对于理解其固有的生物学作用和设计基于靶向GQ的控制和操纵功能的策略是重要的。虽然一些生物物理方法已被用来研究GQ的结构和动力学，我们的理解是远远不够的。因此，这项工作探讨了使用定点自旋标记技术，辅以分子动力学模拟，调查GQ构象。一个核苷酸独立的氮氧化物标签（R5），这已经被应用于探测非编码RNA和DNA双链体的构象，连接到多个位点的22个核苷酸的DNA链来自人类端粒序列（hTel-22），这是已知的形成GQ。显示R5标签对GQ折叠的影响最小，并且显示使用双电子-电子共振光谱法测量的R5间距离足以区分hTel-22的不同拓扑构象，并且响应于环境变量（例如作为盐、拥挤剂和小分子配体）的变化而报告它们的占位性的变化。这项工作表明，R5标签能够探测GQ构象，并为使用R5研究更复杂的序列奠定了基础，例如那些可能在长端粒重复序列中形成多聚体GQ的序列。
Guanine-rich oligonucleotides can form a unique G-quadruplex (GQ) structure with stacking units of four guanine bases organized in a plane through Hoogsteen bonding. GQ structures have been detected in vivo and shown to exert their roles in maintaining genome integrity and regulating gene expression. Understanding GQ conformation is important for understanding its inherent biological role and for devising strategies to control and manipulate functions based on targeting GQ. Although a number of biophysical methods have been used to investigate structure and dynamics of GQs, our understanding is far from complete. As such, this work explores the use of the site-directed spin labeling technique, complemented by molecular dynamics simulations, for investigating GQ conformations. A nucleotide-independent nitroxide label (R5), which has been previously applied for probing conformations of noncoding RNA and DNA duplexes, is attached to multiple sites in a 22-nucleotide DNA strand derived from the human telomeric sequence (hTel-22) that is known to form GQ. The R5 labels are shown to minimally impact GQ folding, and inter-R5 distances measured using double electron–electron resonance spectroscopy are shown to adequately distinguish the different topological conformations of hTel-22 and report variations in their occupancies in response to changes of the environment variables such as salt, crowding agent, and small molecule ligand. The work demonstrates that the R5 label is able to probe GQ conformation and establishes the base for using R5 to study more complex sequences, such as those that may potentially form multimeric GQs in long telomeric repeats.