A nuclear-targeted activity-based sensing probe for ratiometric imaging of formaldehyde reveals endogenous epigenetic contributors to the nuclear formaldehyde pool

Formaldehyde (FA) is both a one-carbon (1C) metabolite and a potent genotoxin in cells. FA plays beneficial roles in endogenous catabolic processes and cellular signaling but its potent electrophilicity necessitates strict regulation. This dichotomy is especially important in the nucleus, where endogenously produced FA has been shown to promote toxicity and disease by generating deleterious DNA adducts. More broadly, the sources and scavenging mechanisms of FA differ across subcellular compartments, underscoring the need for imaging sensors with subcellular resolution to accurately probe contributions of FA to transient, local 1C pools. Here, we report the development of NucRFAP-2, a nuclear-targeted, activity-based ratiometric probe for FA detection, and apply it to monitor dynamic changes in the nuclear FA pool. Using this first-generation reagent for nuclear FA imaging, we demonstrate that genetic perturbation of key FA clearance pathways alters nuclear FA levels by identifying alcohol dehydrogenase 5 (ADH5) as a principal regulator of nuclear FA homeostasis. Furthermore, NucRFAP-2 reveals elevated nuclear FA pools in patient-derived T and B lymphocytes deficient in Wiskott-Aldrich syndrome protein (WASp) and Fanconi anemia group D2 protein (FANCD2), suggesting that replication-associated epigenetic rewiring may contribute to aldehyde-associated pathologies. By demonstrating the ability of NucRFAP-2 to uncover an interplay between FA metabolism, genome integrity, and 1C homeostasis, we showcase this probe as a potentially powerful chemical tool to uncover novel mechanisms of nuclear FA biology.