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

01 October 2021, Version 1
This content is a preprint and has not undergone peer review at the time of posting.

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) dieno-phile. 6-(2-Pyridyl)-dihydrotetrazine-3-carboxamides were developed as stable, cell permeable DHTz reagents that upon oxidation pro-duce the most reactive tetrazines ever used in live cells with Diels-Alder kinetics exceeding k2 106 M-1s-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 was demonstrated. CABL is light-dose dependent, and 2-photon excitation promotes CABL at the sub-organelle level to selectively pattern live cells under no-wash conditions. CABL was also applied to spatially resolved live-cell labeling of an endogenous pro-tein target by using TIRF microscopy to selectively activate intracellular monoacylglycerol lipase tagged with DHTz-labeled small mole-cule 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.

Keywords

photoactivation
bioorthogonal chemistry
photocatalysis
live cell
subcellular
tetrazine
dihydrotetrazine
trans-cyclooctene
long wavelength

Supplementary materials

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Supporting Information
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Synthetic procedures and compound characterization data; methods for determining dihydrotetrazine stability and photooxidation kinetics; Diels-Alder kinetics; description of plasmids; protocols for HaloTag conjugation and photoactivation; protocols for photoactivation of DHTz-HaloTag in e. coli; protocols for photoactivation of DHTz-HaloTag in HeLa Cells; protocols for photoactivation of DHTz-MAGL in PC3 Cells.
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Movie File 1
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HeLa cells expressing HaloTag-H2B-mCherry and labelled with DHTz-Halo. Cells were treated with a-TCO-SiR (100 nM) and FDA (10 µM). The area circled in yellow (lower left) was irradiated with 3.3 second pulses of 880 nm light at 10% laser power, and the resulting increase in fluorescence as a-TCO-SiR is recruited to the nascent Tz is clearly visible. (Scale bar=10 µm)
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Movie File 2
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HeLa cells expressing HaloTag-H2B-mCherry and labelled with DHTz-Halo. Cells were treated with a-TCO-SiR (100 nM) and FDA (10 µM). A square patch of a single nucleus is irradiated with a single pulse of 880 nm light at 25% laser power, leading to a rapid recruitment of a-TCO-SiR (Scale bar=10 µm)
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Movie File 3
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HeLa cells expressing HaloTag-H2B-mCherry and labelled with DHTz-Halo. Cells were treated with a-TCO-SiR (100 nM) and FDA (10 µM). An “X” shape irradiated across the entirety of a single cell with a single pulse of 880 nm light at 25% laser power, leading to a rapid recruitment of a-TCO-SiR in the nucleus alone. This demonstrates that no off-target labeling was observed from non-specific binding to proteins not labeled with DHTz-Halo. (Scale bar=10 µm)
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Movie File 4
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A z-stack was generated from the cell shown in Fig. 7D, demonstrating that CABL possesses a high degree of both axial and lateral resolution.
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Movie File 5
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PC3 cells were labeled with probe 14 for one hour, then treated with a-TCO-SiR (100 nM) and FDA (10 µM). The cells were then exposed to pulses of 480 nm light at 2% laser power at regular intervals, demonstrating recruitment of a-TCO-SiR to endogenous MAGL. (Scale bar=10 µm)
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