DNA Framework-Enabled High-Fidelity Mapping of Biothiol Fluxes at Subcellular Spatial Resolution

Activatable small-molecule probes (ASMPs) enable dynamic biomarker visualization, yet nonspecific sequestration in hydrophobic protein microdomains compromises imaging fidelity. To address this, herein, a size-defined tetrahedral DNA framework (TDF) directs the interior encapsulation of an ASMP to fabricate a DNA framework-confined molecular probes (DFP) via programming the orientation of ASMP-DNA conjugate on the edge of TDF. The DFP architecture confines ~ 99.1 % of conformational freedom of ASMP within the DNA framework and sterically excludes biomolecules ≥ ~ 2.0 nm, thus preserving ~ 82.88 % of the intrinsic response sensitivity while enabling specific response toward small-molecule biothiols under physiological environment. With these unparalleled features, cell membrane- and endocytic organelle-targeting DFPs were designed to map the fluxes of small-molecule biothiols with subcellular spatial resolution in multiple hepatoblastoma cells, and chemically resolved the metabolic difference in location-specific small-molecule biothiols among distinct hepatoblastoma subpopulations during ferroptosis execution. This work establishes a universal strategy to mitigate microenvironmental interference, advancing spatially resolved pathophysiological interrogation.