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

Abstract

Formaldehyde (FA) is both a one-carbon (1C) metabolite and a potent genotoxin in living 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 spatial resolution to accurately probe contributions of FA to transient, local 1C pools. Here, we report 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 reveal 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.

Scheme 1. Design and synthesis of Nuclear Ratiometric Formaldehyde Probe-2 (NucRFAP-2), an activity-based sensing probe for nuclear FA detection. (a) The reaction for activity-based sensing of FA occurs via a key 2-aza-Cope rearrangement. (b) Enzymatic sources and clearance mechanisms that contribute to cellular FA pools, as revealed by previous FAP and RFAP derivatives. In the nucleus, several enzymes, including ALKBH5 and LSD1 shown here, catalyze oxidative demethylation of epigenetic methyl marks on RNA and histones, generating FA as a product. Folates in multiple subcellular compartments undergo spontaneous hydrolysis and/or oxidaitive decomposition, releasing FA. In the mitochondria, ALDH2 functions as a mitochondrial-specific FA scavenger, directly oxidizing FA to form formate. In the cytosol, ADH5, is considered the principal FA detoxifying enzyme. Notably, ADH5 deficiency has been shown to induce 1C modifications on DNA nucleobases, suggesting functional crosstalk between nuclear and cytoplasmic FA pools. Finally, serine cleavage by SHMT2 has been demonstrated to release FA in live cell mitochondria. (c) Synthetic scheme for NucRFAP-2, linking an activity-based sensing dye for FA detection with a Hoechst nuclear targeting moiety.

Fig. 1. In vitro characterization of NucRFAP-2. (a) Binding curve for NucRFAP-2 to an A/T rich DNA hairpin, validating its nanomolar binding affinity. Error bars represent SEM, n = 3. (b) Change in excitation spectrum of NucRFAP-2 (10 µM) in response to FA (100 µM) when bound to DNA hairpin (50 µM) in PBS (1% DMSO). Excitation spectra shown at 0, 15, 30 45, 60, 90, and 120 min (blue, red, green, pink, orange, black, brown). (c) Quantification of the ratio of emission (λ_em = 510 nm) at λ_ex = 470 nm (product) and λ_ex = 420 nm (reactant) of NucRFAP-2 (10 µM) in response to FA (100 µM) over a 2 h incubation period in the presence of vehicle (gray) or DNA hairpin (50 µM, red) in PBS (1% DMSO). Error bars represent SEM, n = 3. (d) Ratiometric response of NucRFAP-2 (10 µM) exposed to several biological analytes (100 µM, unless otherwise noted) in the presence of hairpin DNA (50 µM); measurements were made at 0, 30, 60, 90, and 120 min. (1) PBS; (2) FA; (3) acetaldehyde; (4) methylglyoxal; (5) glutathione, 1 mM; (6) H₂O₂; (7) alpha-keto glutarate; (8) oxaloacetate; (9) pyruvate. Error bars represent SEM, n = 2.

Fig. 2. NucRFAP-2 localizes to live cell nuclei and reports on exogenous additions of FA. (a) Validation of the nuclear localization of NucRFAP-2. HEK-293 FlpIn cells stably expressing Halo-SNAP protein in the nucleus or cytosol, stained with SiR-Halo (500 nM, λ_ex = 633 nm) and NucRFAP-2 (2 µM) for 1 h followed by incubation in HBSS (+0.5% DMSO), for 0.5 h before imaging. Quantitation of overlap of the staining by SiR-Halo and NucRFAP-2 by calculation of Pearson's correlation coefficient. Scale bar represents 25 µm, error bars denote SEM, n = 7 individual cells. (b) Detection of exogenous FA addition with NucRFAP-2. Live HEK-293T cells were stained with NucRFAP-2 (2 µM) for 0.5 h in HBSS (+0.5% DMSO), followed by incubation with 0, 125, 250 or 500 µM FA in HBSS for 1 h. Quantification of the FA-dose dependent, ratiometric response of NucRFAP-2 with psuedocolor to represent the fluorescence emission at λ_ex = 488 nm/λ_ex = 405 nm. Scale bar represents 50 µm; error bars denote SEM, n = 4. Statistical test is an unpaired t test, ****P < 0.0001.

Fig. 3. Detection of intranuclear FA elevation in ADH5 KO MEF cells with NucRFAP-2. (a) Live wild-type MEF stained with NucRFAP-2 (2 µM) for 0.5 h in HBSS followed by treatment with vehicle or FA (62.5 µM) for 1 h. Scale bar represents 50 µm. (b) Live wild-type or ADH5 KO MEF cells were treated with NucRFAP-2 (2 µM) for 1 h in HBSS followed by incubation in full media (high glucose DMEM +10% FBS) for 3 h with vehicle or FA (125 µM). Quantification of the ratiometric response of NucRFAP-2, λ_ex = 488 nm/λ_ex = 405 nm, via flow cytometry. Error bars denote SEM, n = 4. Statistical test is an unpaired t test, ****P < 0.0001. (c) Live wild-type or ADH5 KO MEF cells were treated with NucRFAP-2 (2 µM) for 1 h followed by incubation in full media (DMEM +10% FBS) for 0, 2, 4, 6, 8, 11 or 23 additional h. Fluorescence measurements made via flow cytometry, λ_ex = 488 nm/λ_ex = 405 nm is presented here.

Fig. 4. Detection of intranuclear FA during replication stress in T and B cells by NucRFAP-2. (a) Live human CD4⁺ T cells treated with exogenous FA (500 µM) and stained with NucRFAP-2 (2.5 µM). Images were acquired at λ_ex = 405 nm and λ_ex = 488 nm. Shown are collapsed composite overlays of DIC and fluorescence channels, demonstrating nuclear localization of the signal. Scale bar represents 2.5 µm. (b) Quantification of dose-dependent NucRFAP-2 response to exogenous FA in wild-type T cells. Psuedo color represents the emission at λ_ex = 488 nm/λ_ex = 405 nm. Error bars represent SEM, n = 3. (c) Wild-type, WASp-deficient, and FANCD2-deficient T cell lines treated with hydroxyurea (HU, 200 µM) or vehicle control, stained with NucRFAP-2, and imaged. WASp- and FANCD2-deficient T cell lines were generated by CRISPR/Cas9 knockout of the respective genes in wild-type T cells. Error bars represent SEM, n = 4. (d) Wild-type, WASp-deficient, and FANCD2-deficient patient derived B cell lines treated with hydroxyurea (HU, 200 µM) or vehicle control, stained with NucRFAP-2, and imaged. Patient-derived WAS and Fanc D2 mutant B cell lines carried germline mutations in WAS or FANCD2. Psuedocolor represents the emission at λ_ex = 488 nm/λ_ex = 405. Scale bar represents 10 µm. Quantification of HU-induced intranuclear FA accumulation in the indicated T and B cell lines. Error bars represent SEM, n = 12. Statistical test is an unpaired t test, *P < 0.05 **P < 0.01 ***P < 0.001 ****P < 0.0001.