Improving the robustness of a catalyzed hairpin assembly with a three-arm nanostructure for nonenzymatic signal amplification

Kinetically trapped hairpin DNA has great potential to dynamically build nanostructures, which can be initiated by sequence-specific nucleic acids. The branched junction, which has a multi-arm structure, is a representative nanostructure of DNA. In this study, we report a nonenzymatic and isothermal signal amplification accompanied by building a 3-arm structure based on a catalyzed hairpin DNA assembly (3-CHA). We improved the signal-to-background ratio of the 3-CHA by suppressing the leakage pathway of 3-CHA, thus eliminating unfavorable reaction sites exposed in the single-stranded region of hairpin DNAs. Background and amplified signals were analyzed with gel electrophoresis and real-time fluorescence monitoring. The limit of detection of the developed 3-CHA was estimated to be 29.3 pM for catalyst DNA at room temperature. Supported by the reduced leakage signal, the implemented 3-CHA showed great potential for detecting low concentrations of target DNA.