Exploring Base-Stacking Interactions to Energize Kinetic and Amplifiable Catalytic Hairpin Assembly Circuits

Abstract In typical catalytic hairpin assembly (CHA) mediated by long toeholds, the hard balance between the reaction rate and nonspecific background needs to be addressed. Herein, we propose the first example of a dinucleotide base-stacking-energized CHA (bsCHA) strategy by shortening the sticky toehold of a reactive hairpin. To offset the retarded reaction speed, the coaxial adenine|guanine (A|G) stacking is leveraged as a driving energy for operating bsCHA. In our design, a functional bihairpin (fbH) is programmed with two hairpin subunits, and one of them is purposely terminated with a G nucleotide in the 3′-blunt end. A toehold-truncated hairpin (ttH) ending with an A nucleotide is introduced for minimizing unintended background leakage. In a trigger-responsive route, the specific binding with fbH induces the hairpin-structured conformation collapse to liberate its blocked stem. The 5′-toehold of ttH is therefore drawn to dock at fbH for forming an adjacent A|G stack at the right nick site. The resulting A|G interaction offers a useful force to stabilize two-toehold docking for facilitating the ttH unfolding, where the trigger is displaced and detached to energize repetitive cycling events forward, generating numerous highly ordered products. During this progressive bsCHA circuit, the base-stacking interaction enables the offset of reaction kinetics and efficient amplification to implement downstream signal switching by directional strand migration. The simplicity and effectiveness of our bsCHA-based approach provide a fascinating pathway for advancing the applications of DNA catalytic assemblies in biosensing, cell imaging, or logic computation.