An Intelligent Redox‐Responsive DNA Circuit for Robust On‐Site Profiling of Glutathione‐MicroRNA Signaling Pathway

Abstract

Synthetic biochemical circuits (e.g., DNA circuits) remain at the forefront of intracellular biosensing tasks yet are hindered by the undesired off‐site activation and the accompanying signal leakage. Herein, the study attempts to overcome this limitation by developing a simple‐yet‐powerful endogenous glutathione (GSH)‐regulating tactic that permits robust and distinguishable on‐site microRNA (miRNA) imaging under disturbed redox homeostasis. Specifically, a hierarchically activated catalytic DNA (HAD) circuit is fabricated by grafting the disulfide linkage within entropy‐driven DNA circuitry (EDC) reactants. It is exemplified that this HAD system promises the spatiotemporally selective microRNA‐21 (miR‐21) imaging in living cells and the robust differentiation of tumor cells from normal cells. The correlationship between GSH and miRNA is extensively explored in live cells, and can substantially expand the toolbox of DNA circuits for profiling intracellular biochemical processes.

Endogenous Glutathione-Activated Nucleic Acid Molecular Circuitry for Cell-Specific MicroRNA Imaging

“…We selected three types of cells with varying expression content of GSH and miR-21 to investigate the miRNA imaging property of the HCR circuit (Figure A). These cells included MCF-7 cells with high expression of miR-21 and GSH, human cervical cancer cells (HeLa cells) with medium expression of miR-21 and GSH, and human breast normal cells (MCF-10A cells) with relatively low expression of miR-21 and GSH . As illustrated in Figure B (for details, see Figure S12), distinct fluorescence signals were captured in tumorous cell lines (MCF-7 cells and HeLa cells) incubated with the GSH-activated HCR circuit (sample a and sample b, respectively).…”

“…These cells included MCF-7 cells with high expression of miR-21 and GSH, human cervical cancer cells (HeLa cells) with medium expression of miR-21 and GSH, and human breast normal cells (MCF-10A cells) with relatively low expression of miR-21 and GSH. 52 As illustrated in Figure 5B (for details, see Figure S12), distinct fluorescence signals were captured in tumorous cell lines (MCF-7 cells and HeLa cells) incubated with the GSHactivated HCR circuit (sample a and sample b, respectively). In contrast, the fluorescence of normal cells (MCF-10A cells) was much weaker (sample c).…”

A Methylation-Gated DNAzyme Circuit for Spatially Controlled Imaging of MicroRNA in Cells and Animals

“…25−27 The small molecule-based regulation strategies were constrained by the insufficient aptamers with low binding affinities. 21 Meanwhile, the RNA regulation methods are confronted with inefficient regulation capacities as a result of their low expressions in live cells. 28 The enzyme is thus considered as a more promising circuitregulating candidate due to its excellent catalytic activity and biosafety.…”

“…As a typical artificial molecular self-assembly network, − DNA circuits have been utilized for intelligent biosensing and biocomputing applications − based on their intrinsic multiply guaranteed and amplified molecular recognitions. − To eliminate the undesirable disturbance from a surrounding complex biological environment, the endogenously stimulated DNA circuits are thus developed for achieving the site-specific circuitry activation with high spatiotemporal controllability, − and these include small molecules (e.g., ATP), , RNAs, − and enzymes. − The small molecule-based regulation strategies were constrained by the insufficient aptamers with low binding affinities . Meanwhile, the RNA regulation methods are confronted with inefficient regulation capacities as a result of their low expressions in live cells .…”