Construction of an exogenously and endogenously Co-activated DNA logic amplifier for highly reliable intracellular MicroRNA imaging.

DNA-based molecular amplifiers offer significant promise for molecular-level disease diagnosis and treatment, yet tailoring their activation for precise timing and localization remains a challenge. Herein, we've pioneered a dual activation strategy harnessing external light and internal ATP to create a highly controlled DNA logic amplifier (FDLA) for accurate miRNA monitoring in cancer cells. The FDLA was constructed by tethered the two functionalized catalytic hairpin assembly (CHA) hairpin modules (ATP aptamer sealed hairpin aH1 and photocleavable (PC-linker) sites modified hairpin pH2) to DNA tetrahedron (DTN). The FDLA system incorporates ATP aptamers and PC-linkers as logic control units, allowing them to respond to both exogenous UV light and endogenous ATP present within cancer cells. This response triggers the release of CHA hairpin modules, enabling amplified FRET miRNA imaging through an AND-AND gate. The DTN structure could improve the stability of FDLA and accelerate the kinetics of the strand displacement reaction. It is noteworthy that the UV and ATP co-gated DNA circuit can control the DNA bio-computing at specific time and location, offering spatial and temporal capabilities that can be harnessed for miRNA imaging. Furthermore, the miRNA-sensing FDLA amplifier demonstrates reliable imaging of intracellular miRNA with minimal background noise and false-positive signals. This highlights the feasibility of utilizing both exogenous and endogenous regulatory strategies to achieve spatial and temporal control of DNA molecular circuits within living cancer cells. Such advancements hold immense potential for unraveling the correlation between miRNA and associated diseases.