Monitoring G-quadruplex formation with DNA carriers and solid-state nanopores
Monitoring G-quadruplex formation with DNA carriers and solid-state nanopores
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DOI：
10.1101/788513
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发表时间：
2019-10
期刊：
影响因子：
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通讯作者：
F. Bošković;Jinbo Zhu;Kaikai Chen;U. Keyser
中科院分区：
文献类型：
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作者：
F. Bošković;Jinbo Zhu;Kaikai Chen;U. Keyser
g -四plex （Gq）是由单链DNA形成的富含鸟嘌呤的DNA结构。它们对基因表达调控具有重要意义，也是癌症和人类病毒的药物靶点。目前的集合和单分子方法需要荧光标记，这可能会影响Gq折叠动力学。在这里，我们介绍了一种单分子Gq纳米孔测定法（smGNA）来检测Gq和Gq形成的动力学。我们使用~5 nm的固态纳米孔来检测附着在设计的DNA载体上的各种Gq结构变体。Gq可以通过定位它们在设计的DNA载体上的位置来识别，建立smGNA作为Gq定位的工具。此外，smGNA允许辨别（非）折叠的Gq结构，提供对g -四联折叠的单分子动力学的见解，并探测四联到双联的结构转变。smGNA可以在单分子水平上阐明g -四联体的形成而不需要标记，并且对单链DNA和基因组样本中这些结构的研究具有潜在的意义。
G-quadruplexes (Gq) are guanine-rich DNA structures formed by single-stranded DNA. They are of paramount significance to gene expression regulation, but also drug targets for cancer and human viruses. Current ensemble and single-molecule methods require fluorescent labels, which can affect Gq folding kinetics. Here we introduce, a single-molecule Gq nanopore assay (smGNA) to detect Gqs and kinetics of Gq formation. We use ~5 nm solid-state nanopores to detect various Gq structural variants attached to designed DNA carriers. Gqs can be identified by localizing their positions along designed DNA carriers establishing smGNA as a tool for Gq mapping. In addition, smGNA allows for discrimination of (un-)folded Gq structures, provides insights into single-molecule kinetics of G-quadruplex folding, and probes quadruplex-to-duplex structural transitions. smGNA can elucidate the formation of G-quadruplexes at the single-molecule level without labelling and has potential implications on the study of these structures both in single-stranded DNA and in genomic samples.