Single-molecule detection of folding and unfolding of the G-quadruplex aptamer in a nanopore nanocavity

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.