Guanine-rich nucleic acids can form G-quadruplexes that are important in gene regulation, biosensor design and nano-structure construction. In this article, we report on the development of a nanopore encapsulating single-molecule method for exploring how cations regulate the folding and unfolding of the G-quadruplex formed by the thrombin-binding aptamer (TBA, GGTTGGTGTGGTTGG). The signature blocks in the nanopore revealed that the G-quadruplex formation is cation-selective. The selectivity sequence is K+ > NH4+ ∼ Ba2+ > Cs+ ∼ Na+ > Li+, and G-quadruplex was not detected in Mg2+ and Ca2+. Ba2+ can form a long-lived G-quadruplex with TBA. However, the capability is affected by the cation–DNA interaction. The cation-selective formation of the G-quadruplex is correlated with the G-quadruplex volume, which varies with cation species. The high formation capability of the K+-induced G-quadruplex is contributed largely by the slow unfolding reaction. Although the Na+- and Li+-quadruplexes feature similar equilibrium properties, they undergo radically different pathways. The Na+-quadruplex folds and unfolds most rapidly, while the Li+-quadruplex performs both reactions at the slowest rates. Understanding these ion-regulated properties of oligonucleotides is beneficial for constructing fine-tuned biosensors and nano-structures. The methodology in this work can be used for studying other quadruplexes and protein–aptamer interactions.
展开▼
机译:富含鸟嘌呤的核酸可以形成G-四链体,在基因调控,生物传感器设计和纳米结构构建中很重要。在本文中,我们报告了一种纳米孔封装单分子方法的发展,该方法用于探索阳离子如何调节凝血酶结合适体(TBA,GGGTGGTGTGGTTGG)形成的G四联体的折叠和解折叠。纳米孔中的特征性嵌段揭示了G-四链体的形成是阳离子选择性的。选择性顺序为K +> NH4 +〜Ba2 +> Cs +〜Na +> Li +,并且在Mg2 +和Ca2 +中未检测到G-四链体。 Ba2 +可以与TBA形成长寿的G四联体。但是,该能力受阳离子与DNA相互作用的影响。 G-四链体的阳离子选择性形成与G-四链体的体积相关,G-四链体的体积随阳离子种类而变化。 K +诱导的G-四链体的高形成能力在很大程度上是由缓慢的展开反应引起的。尽管Na +-和Li +-四链体具有相似的平衡性质,但它们经历了截然不同的途径。 Na +-四链体折叠和展开最快,而Li +-四链体以最慢的速度进行两种反应。了解寡核苷酸的这些离子调节特性对构建微调的生物传感器和纳米结构是有益的。这项工作中的方法可用于研究其他四链体和蛋白质-适体相互作用。
展开▼