Epigenetic Control of Toehold-Mediated Strand Displacement for Programmable Molecular Circuit Regulation and Enhanced microRNA Detection

Conventional toehold-mediated strand displacement─a cornerstone of dynamic DNA nanotechnology─is fundamentally limited by its reliance on the fixed toehold stability to control reaction kinetics, restricting precise and reversible regulation of molecular circuits. Here, we describe an epigenetically regulated system that exploits single-nucleobase N⁶-methyladenosine (m⁶A) modification within the toehold domain to achieve programmable kinetic control over DNA strand displacement. Site-specific m⁶A methylation disrupts base pairing at the toehold domain, effectively inhibiting strand displacement rates. This inhibition is precisely and reversibly modulated by demethylase FTO, which removes m⁶A modifications and restores toehold reactivity. Applied to catalytic hairpin assembly, this strategy not only enables the controlled inhibition and restoration of nucleic acid circuit function but also enhances its sensitivity and specificity in microRNA detection, exemplified by the intracellular imaging of cancer-associated microRNA-21. Our combined theoretical and experimental analyses indicate that both the location and density of m⁶A modifications critically dictate the extent of reaction inhibition, supporting the use of single-base epigenetic modifications as a versatile tool for chemical system design. This epigenetically regulated platform provides a general framework for dynamic nucleic acid circuits, with broad implications for biosensing, molecular computing, and synthetic biology, advancing the development of epigenetically controlled biochemical systems.