Programmable DNA-Based NIR-II Molecular Probes for Hepatotoxicity-Associated MicroRNA Imaging <i>In Vivo</i>

DNA-based molecular probes are powerful, programmable tools for molecular sensing but are largely confined to the visible spectrum, where poor tissue penetration obstructs their deep-tissue imaging applications. While NIR-II fluorescence offers profound advantages for deep-tissue imaging, the transition of these probes into this optimal window is obstructed by the scarcity of high-performance fluorophore-quencher combinations. Here, we introduce a synergistic design strategy that overcomes this limitation by coengineering a high-brightness NIR-II emitter and its spectrum-matched quencher. We first created the high-brightness NIR-II emitter by stoichiometrically conjugating a DNA strand and a cyanine dye to an albumin scaffold. This encapsulation generates a stable and bright NIR-II tail emission with an NIR-II photoluminescence quantum yield 10-fold higher than that of commercial NIR-II dyes, enabling tissue penetration beyond 8 mm. We then rationally design the quencher via a symmetry-breaking strategy on the cognate chromophore, a one-step conversion that precisely aligns its absorption with the emitter's peak emission. This approach yields the DAC-783/Q783 combination, which exhibits a significantly higher quenching efficiency than a conventional tail-emission-matched quencher and enables robust DNA-based NIR-II molecular probes (NMPs). As a proof of concept, a probe targeting microRNA-122 (NMP-122) significantly outperforms conventional visible-window probes in deep tissue and enables noninvasive, real-time monitoring of drug-induced liver injury in living mice. This work extends the operational window of molecular probes into the NIR-II region for the first time and, more broadly, establishes a validated and generalizable blueprint for codesigning custom-designed fluorophore-quencher combinations, paving a new pathway to the clinical translation of NIR-II DNA nanotechnology.