Resolution Enhancement of Nanopore Molecular Assay Based on the Repeating Sequences-Duplex Polymerization Strategy, a Kind of “Self-Tag” Strategy

Nanopore analysis is one of the most promising single-molecule technology. For molecular detection, the duplex polymerization strategy (DPS) serves as an effective signal amplification and conversion approach. However, it faces challenges including low ligation efficiency, difficulty in distinguishing products with multiple lengths, high cost, and complex synthesis steps for generating secondary peaks. To address these issues, we develop an enhanced strategy termed the repeating sequences-duplex polymerization strategy (RS-DPS). The strategy increases the number of structural domains in the linkers, providing more binding sites to improve the ligation efficiency. The framework generates secondary peaks by self-labeling, instead of a traditional "tag-on" step utilizing nanostructures such as DNA tetrahedrons, reducing costs and simplifying experimental procedure. Additionally, RS-DPS is advantageous in terms of universality, modularity, and editability. Using APOE genotyping as a proof of concept, we employ two methods to achieve the multiplex detection of two duplex substrate (DS) types. Under the RS-DPS strategy, two target sites can generate target-specific DS homopolymers with two kinds of linkers, respectively. Furthermore, two kinds of DS homopolymers can be distinguished through nanopore signal density plots supplemented by single molecule signal features (<i>I</i>₁/<i>I</i>₀). Finally, to meet multiplex detection requirements, we used a single linker to simultaneously detect two DS types. By ligating two types of DS, distinct DS polymers are formed depending on their different coexistence situations, enabling multiplex detection of the two DS types through nanopore signal density plots and single-molecule <i>I</i>₁/<i>I</i>₀ signals.