Computationally steered toehold engineering accelerates primer exchange reaction for rapid detection of congenital heart disease-associated miRNAs.

Abstract

Isothermal nucleic acid amplification, including the primer exchange reaction (PER), provides a promising basis for molecular diagnostics with potential for point-of-care testing (POCT). However, its clinical translation is severely bottlenecked by prolonged reaction times (typically >4 h) originating from the intrinsic nucleation barrier during initial target recognition. Here, we propose a synergistic computational-experimental paradigm to accelerate PER kinetics by rationally modulating the target-recognition toehold length. By employing oxDNA coarse-grained molecular dynamics simulations and NUPACK thermodynamic computations, we systematically decode the free-energy landscape of target-probe interactions. Both theoretical modeling and experimental validation consistently reveal that a precisely extended 15-nucleotide (nt) recognition toehold facilitates initial target recognition and promotes subsequent strand displacement, thereby accelerating the initiation of the PER process. Operating via a programmable dGTP-depleted enzymatic cascade, this precision molecular engineering yields a 75% temporal reduction, compressing PER amplification time to a mere 60 min. The toehold-engineered amplification platform achieves an analytical limit of detection of approximately 662 fM, demonstrating its capability for low-concentration target detection. The observed differences in fluorescence responses among the clinical groups suggest the feasibility of applying the platform to CHD-associated circulating miRNA profiling. We further evaluated the accelerated PER platform by separately detecting three CHD-associated miRNAs (miR-146a-5p, miR-122-5p, and miR-548am-3p) in clinical serum samples and combining their fluorescence responses for exploratory analysis. Preliminary differences were observed among the clinically defined groups. This study establishes a thermodynamically steered kinetic acceleration strategy for dynamic DNA reactions, offering a rapid, reliable, and clinically actionable avenue for early disease screening.

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