Lego‐Like Catalytic Hairpin Assembly Enables Controllable DNA‐Oligomer Formation and Spatiotemporal Amplification in Single Molecular Signaling

Abstract While the solid‐state nanopore shows increasing potential during sensitive and label‐free single molecular analysis, target concentration and signal amplification method is in urgent need. In this article, a solution via designing a model nucleic acid circuit reaction that can produce “Y” shape‐structure three‐way DNA oligomers with controllable size and polymerization degree is proposed. Such a so‐called lego‐like three‐way catalytic hairpin assembly (LK‐3W‐CHA) can provide both concentration amplification (via CHA circuit) and programmable size control (via lego‐like building mode) to enhance spatiotemporal resolution in single molecular sensing of solid‐state nanopore. Oligomers containing 1–4 DNA three‐way junctions (Y monomers, Y1–Y4) are designed in proof‐of‐concept experiments and applications. When the oligomers are applied to direct translocation measurements, Y2‐Y4 can significantly increase the signal resolution and stability than that of Y1. Meanwhile, Y1 to Y4 can be used as the tags on the long DNA carrier to provide very legible secondary signals for specific identification, multiple assays, and information storage. Compared with other possible tags, Y1–Y4 provides higher signal density and amplitude, and quasi‐linear “inner reference” for each other, which may provide more systematic, reliable, and controllable experimental results.