Breaking the stoichiometric limit of padlock probe ligation via catalytic hairpin assembly-and-cyclization.

Rolling circle amplification (RCA) is a powerful isothermal nucleic acid amplification technique prized for its robustness and simplicity. However, conventional RCA-based detection is fundamentally limited by the stoichiometry of padlock probe ligation, wherein each target molecule ideally yields only one circular template. Existing strategies to improve ligation efficiency often sacrifice key benefits of RCA or focus on specificity rather than catalytic turnover. Herein, we developed catalytic hairpin assembly-and-cyclization (CHAC), a homogeneous cascade that integrates catalytic hairpin assembly with enzymatic ligation, enabling each target to initiate multiple circularizations and breaking the 1:1 stoichiometric limit. As the target serves only to initiate catalytic assembly without acting as the substrate for ligation or the primer for RCA, CHAC overcomes constraints on target identity and topology. Applied to detection of the mpox (monkeypox) virus E9L gene using a single-tube format (incorporating detection probe-modified magnetic nanoparticles) with real-time optomagnetic sensing, CHAC-RCA achieved detection limits of 0.3-2 fM (depending on amplification duration) within a total assay time of ~2-2.5 h, representing at least a 100-fold improvement over conventional ligation-RCA. The assay showed high specificity, robustness, and clinical concordance with quantitative PCR, establishing CHAC-RCA as a versatile and efficient platform for ultrasensitive nucleic acid detection.