Catalytic Activation of Bioorthogonal Chemistry with Light (CABL) Enables Rapid, Spatiotemporally Controlled Labeling and No-Wash, Subcellular 3D-Patterning in Live Cells Using Long Wavelength Light

Abstract

Described is the spatiotemporally controlled labeling and patterning of biomolecules in live cells through the catalytic activation of bioorthogonal chemistry with light, referred to as "CABL". Here, an unreactive dihydrotetrazine (DHTz) is photocatalytically oxidized in the intracellular environment by ambient O₂ to produce a tetrazine that immediately reacts with a trans-cyclooctene (TCO) dienophile. 6-(2-Pyridyl)dihydrotetrazine-3-carboxamides were developed as stable, cell permeable DHTz reagents that upon oxidation produce the most reactive tetrazines ever used in live cells with Diels-Alder kinetics exceeding k₂ of 10⁶ M^-1 s^-1. CABL photocatalysts are based on fluorescein or silarhodamine dyes with activation at 470 or 660 nm. Strategies for limiting extracellular production of singlet oxygen are described that increase the cytocompatibility of photocatalysis. The HaloTag self-labeling platform was used to introduce DHTz tags to proteins localized in the nucleus, mitochondria, actin, or cytoplasm, and high-yielding subcellular activation and labeling with a TCO-fluorophore were demonstrated. CABL is light-dose dependent, and two-photon excitation promotes CABL at the suborganelle level to selectively pattern live cells under no-wash conditions. CABL was also applied to spatially resolved live-cell labeling of an endogenous protein target by using TIRF microscopy to selectively activate intracellular monoacylglycerol lipase tagged with DHTz-labeled small molecule covalent inhibitor. Beyond spatiotemporally controlled labeling, CABL also improves the efficiency of "ordinary" tetrazine ligations by rescuing the reactivity of commonly used 3-aryl-6-methyltetrazine reporters that become partially reduced to DHTzs inside cells. The spatiotemporal control and fast rates of photoactivation and labeling of CABL should enable a range of biomolecular labeling applications in living systems.

Fig 1.

Illustration of spatiotemporally controlled subcellular labeling via catalytic activation of bioorthogonal chemistry with light, or “CABL”. (A) Directing a DHTz-functionalized ligand to a subcellular target (illustrated here for actin) does not result in recruitment of a labeled TCO until (B) illumination in the presence of light results in the oxidation of DHTz to tetrazine, enabling rapid labeling via the fastest bioorthogonal reactions observed to date in live cells.

Fig 2.

(A) Silarhodamine or fluorescein dyes catalyze the oxidation of dihydrotetrazine (DHTz) 1 by O₂ to produce tetrazine 2. (B) Synthesis of amine-reactive DHTz 6 and amide analogs 1a-c. (C) Relative rate of tetrazine-TCO ligation for 2a vs other tetrazine derivatives in aqueous buffer. (D) Kinetics for reaction of 2a with TCO derivatives.

Fig 3

(A) DHTz oxidation/Diels-Alder reaction of 1a with BCN is photocatalyzed by either SiR or fluorescein gives conjugate 10. (B) Photocatalytic DHTz oxidation/Diels-Alder reaction of Halo-DHTz in the presence of methionine, a singlet oxygen scavenger, produces conjugate 11.

Fig 4

(A) Workflow for introducing DHTz labels to Halotag fusion proteins in HeLa cells or E. coli with subsequent fluorescent labeling via CABL. (B) Structure of TAMRA-TCO conjugates. (C) Upon hydrolysis by esterases, fluoresein diacetate becomes fluorescent and an active photocatalyst in live cells. (D, E) In-gel fluorescence was used to monitor the progress of subcellular photocatalysis using (D) Fluorescein/470 nm light/60 mW/cm² and (E) SiR-tJF₆₄₆/660 nm/450 mW/cm² light.

Fig 5.

CABL improves the efficiency of a ‘regular’ bioorthogonal tetrazine ligation. (A) Intracellular labeling of a MeTzHalo-tagged protein proceeds with low labeling efficiency relative to direct labeling by a (C) TAMRA-halo control. (B) The low efficiency of the tetrazine ligation was hypothesized to be a result of inactivation of the tetrazine in the cellular environment. (D) UV-vis spectroscopic monitoring of Tz 9 in PBS containing 5 mM glutathione in the presence of SiR shows that tetrazine absorption slowly decreases in the dark, rapidly recovers upon illumination, and again decreases in the dark. (E) The efficiency of tetrazine ligation with MeTzHalo in live cells is improved 3.6-fold through photocatalysis and is more comparable to that observed when HeLa cells expressing Halo-Tag-H2B-GFP were directly tagged with TAMRA-Halo (Fig 5E, right).

Fig 6.

Fluorescence imaging of subcellular targets in live cells labeled by CABL. (A) DHTz-HaloTag is introduced to HeLa cells were transfected with a HaloTag-POI-GFP fusion where the protein of interest (POI) controls subcellular localization. Successful photoactivation and Diels-Alder reaction with TAMRA-TCO results in the colocalization of green fluorescence from the target and red fluorescence from TAMRA. Cells were labeled while live and fixed prior to imaging, and DAPI was added to stain cell nuclei. (B) Confocal fluorescence microscopy images of photoactivate cells with DHTz targeted to LifeAct (actin), GAP43 (cytoplasm), ActA (mitochondria) and H2B (nucleus). Scale bar = 10 μm

Fig 7.

Live cell, no-wash photopatterning on the cell nucleus. (A) In the presence of a-TCO-SiR, two-photon excitation microscopy (880 nm) is used to activate fluorescein and photocatalyze the generation of reactive tetrazines in nuclei of HaloTag-H2B-RFP transfected cells. The whole nucleus is red-fluorescent, but only the photopatterned regions are labeled by the far-red SiR-dye. Cells are imaged live immediately after 2-photon excitation. (B-D) Illuminating for 3.3 seconds with focused, 2-photon light is used to label (B) square, (C) an ‘X’ and (D) letters in the cell nucleus. A high laser power (details) was used in (C) to demonstrate that labeling is effective even under conditions that photobleach the RFP fluorophore. (E) For the experiment in Fig 7B, the intensity of fluorescence at 633 nm due to the SiR fluorophore was monitored in the illuminated region of the nucleus as well as in a non-illuminated square region of equivalent area found directly below. (F). Fluorescence intensity timecourse for a cell nucleus of a single cell that was periodically pulsed with light from the 2-photon source at low laser power. The fluorescence threshold was set to the background fluorescence of the SiR dye, and detection above the threshold is displayed. After irradiation, the fluorescence intensity is approximately an order of magnitude higher in the irradiated nucleus relative to the nucleus of a neighboring nucleus. Scale bar = 10 μm

Fig 8.

(A) Activity-based labeling of endogenous MAGL in live cells followed by photocatalytic oxidation (B) Structure of MAGL reactive probe 14 and competitive inhibitor KML-29. (C) Live cells were treated with probe 14 for 1 h, washed and then treated with FDA (10 μM) for 30 min, followed by 2 μM a-TCO-SiR for 30 min, and irradiation for 1 min with 470 nm light. A non-fluorescent tetrazine was added to quench unreacted a-TCO-SiR, and cells were lysed and analyzed by in-gel fluorescence. (C) In-gel fluorescence signals for a dose response of probe 14. (D) Dose response fitting of the fluorescence signals of MAGL normalized by the total protein amount indicated by Coomassie staining. Data are reported as mean ± SEM (n = 2). (E) Probe 14 (3.2 nM, 1 h) was competed by pre-treatment with MAGL inhibitor KML29 (300 nM, 1 h). Additional controls include the exclusion of FDA photocatalyst, light or both light and photocatalyst. See Fig S25 for Coomassie staining. (F-I) Confocal microscopy data for live PC3 cells that were labeled by 14 and incubated with a-TCO-SiR before and after wide field illumination with 470 nm light. The fluorescence threshold was set to the background fluorescence of the SiR dye, and detection above the threshold is displayed. (F) SiR fluorescence + DIC prior to illumination. (G) SiR fluorescence + DIC after illumination. (H) SiR fluorescence after illumination. (I) Fluorescence intensity timecourse monitoring of a region of a cell that was periodically pulsed with 488 nm light (Scale bar=10 μm).

Fig 9.

(A) Total internal reflection fluorescence (TIRF) microscopy was used to activate spatially resolved live-cell labeling of endogenous MAGL proteins that were covalently labeled by probe 14. Only protein-DHTz conjugates in the thin region of evanescent illumination become activated and labeled by the a-TCO-SiR fluorophore. (B) Top view of illuminated and non-illuminated cells visualized by fluorescence microscopy. (C,D) Perspective and orthogonal view of illuminated cells visualized by microscopy. In the orthogonal view, the arrow points to the thin layer that becomes fluorescently labeled near the glass surface. (E) Plot of fluorescent intensity vs distance from surface for the orthogonal projection.