Enzymatic Ligation-Enhanced Catalytic Hairpin Assembly for Accelerated Kinetics with Restricted Leakage

Abstract

Enthalpy-driven catalytic hairpin assembly (CHA) suffers from an inherent trade-off, wherein enhanced reaction kinetics is inevitably accompanied by elevated nonspecific background. Catalytic hairpin assembly-and-cyclization (CHAC) integrates CHA with enzymatic ligation, allowing single-target-triggered multiple circularization events and thus bypassing the stoichiometric limitation of conventional padlock probe ligation. Nevertheless, how the introduction of a ligase reaction remodels the enthalpy-driven DNA circuit remains unclear. Herein, we systematically characterize kinetic profiles and background leakage behaviors of CHAC, confirming that T4 DNA ligase enhances overall product accumulation, with a more pronounced kinetic advantage at moderate to high target concentrations. Notably, the spontaneous upregulation of nonspecific leakage, typically induced alongside kinetic improvement, is effectively restricted in CHAC. Leveraging this enhanced kinetic performance with restricted background leakage, we further combined CHAC with nicking-enhanced rolling circle amplification (NickRCA) to construct an optomagnetic biosensor targeting the mecA gene in methicillin-resistant Staphylococcus aureus. CHAC-derived circular templates guide NickRCA to generate abundant short single-stranded amplicons, which cross-link detection probe-modified magnetic nanoparticles and elicit quantitative optomagnetic responses. This study characterizes ligase-dependent kinetic and leakage modulation of the present CHAC system, with the underlying mechanism proposed to involve both covalent cyclization and noncovalent duplex binding effects, which may be generalizable to other DNA strand displacement circuits.