Iron is essential for sustaining life, as its ability to cycle between multiple oxidation states is critical for catalyzing chemical transformations in biological systems. However, without proper regulation, this same redox capacity can trigger oxidative stress events that contribute to aging along with diseases ranging from cancer to cardiovascular and neurodegenerative disorders. Despite its importance, methods for monitoring biological iron bound weakly to cellular ligands−the labile iron pool−to generate a response that preserves spatial and temporal information remain limited, owing to the potent fluorescence quenching ability of iron. We report the design, synthesis, and biological evaluation of FRET Iron Probe 1 (FIP-1), a reactivity-based probe that enables ratiometric fluorescence imaging of labile iron pools in living systems. Inspired by antimalarial natural products and related therapeutics, FIP-1 links two fluorophores (fluorescein and Cy3) through an Fe(II)-cleavable endoperoxide bridge, where Fe(II)-triggered peroxide cleavage leads to a decrease in fluorescence resonance energy transfer (FRET) from the fluorescein donor to Cy3 acceptor by splitting these two dyes into separate fragments. FIP-1 responds to Fe(II) in aqueous buffer with selectivity over competing metal ions and is capable of detecting changes in labile iron pools within living cells with iron supplementation and/or depletion. Moreover, application of FIP-1 to a model of ferroptosis reveals a change in labile iron pools during this form of cell death, providing a starting point to study iron signaling in living systems.

Chemical modification of nucleic acids in living cells can be sterically hindered by tight packing of bioorthogonal functional groups in chromatin. To address this limitation, we report here a dual enhancement strategy for nucleic acid‐templated reactions utilizing a fluorogenic intercalating agent capable of undergoing inverse electron‐demand Diels–Alder (IEDDA) reactions with DNA containing 5‐vinyl‐2′‐deoxyuridine (VdU) or RNA containing 5‐vinyl‐uridine (VU). Reversible high‐affinity intercalation of a novel acridine–tetrazine conjugate “PINK” (KD=5±1 μM) increases the reaction rate of tetrazine–alkene IEDDA on duplex DNA by 60 000‐fold (590 M−1 s−1) as compared to the non‐templated reaction. At the same time, loss of tetrazine–acridine fluorescence quenching renders the reaction highly fluorogenic and detectable under no‐wash conditions. This strategy enables live‐cell dynamic imaging of acridine‐modified nucleic acids in dividing cells.

Fluorescent nucleoside triphosphates are powerful probes of DNA synthesis, but their potential use in living animals has been previously underexplored. Here, we report the synthesis and characterization of 7-deaza-(1,2,3-triazole)-2′-deoxyadenosine-5′-triphosphate (dATP) derivatives of tetramethyl rhodamine (“TAMRA-dATP”), cyanine (“Cy3-dATP”), and boron-dipyrromethene (“BODIPY-dATP”). Upon microinjection into live zebrafish embryos, all three compounds were incorporated into the DNA of dividing cells; however, their impact on embryonic toxicity was highly variable, depending on the exact structure of the dye. TAMRA-EdATP exhibited superior characteristics in terms of its high brightness, low toxicity, and rapid incorporation and depletion kinetics in both a vertebrate (zebrafish) and a nematode (Caenorhabditis elegans). TAMRA-EdATP allows for unprecedented, real-time visualization of DNA replication and chromosome segregation in vivo.