Catalytic activation of bioorthogonal chemistry with light (CABL) enables rapid spatiotemporally controlled labeling, no-wash subcellular 3D-patterning, and tetrazine-based redox probing in live cells
Date
2023
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
I have spent my time in the Fox lab developing new methodologies and applications for the bioorthogonal reaction between tetrazine (Tz) and trans-cyclooctene (TCO). Specifically, I have been investigating the use of a reduced version of Tz, dihydrotetrazine (DHTz), which is unreactive towards TCO until photooxidized to Tz using photocatalyst and light. DHTz turn-on chemistry has been demonstrated for a number of in vitro applications, so the focus of my work has been on developing this methodology for intracellular application within living cells. ☐ Chapter 1 covers the development of the catalytic activation of bioorthogonal chemistry with light (CABL), wherein DHTz is oxidized to Tz in a cellular context. I begin by describing the selection of suitable photocatalysts, examining DHTz’s ability to serve as a bioorthogonal probe in vitro by covalently conjugating it to the self-labeling HaloTag protein, and my attempts to mitigate singlet oxygen damage. Using bacterial and mammalian cells expressing the HaloTag protein, I found that DHTz was a robust bioorthogonal reporter. After conjugating DHTz to HaloTag within living cells, it readily reacted with TCO-fluorophores after being oxidized to Tz in a variety of subcellular locations. The stability of DHTz was also demonstrated, since the reducing environment of the cell allowed it to remain in its reduced state, even after a full day of incubation. DHTz also served as an effective tool for imaging proteins, with confocal microscopy analysis showing a high signal-to-noise ratio for multiple different organelles. Lastly, the ability of DHTz to be spatiotemporally activated on-demand allowed for the subcellular 3D patterning of live cell nuclei using long-wavelength multiphoton light. ☐ In chapter 2, I discuss the development of covalent DHTz probes for endogenous proteins. In collaboration with Pfizer, we created a probe for the cancer protein monoacylglycerol lipase (MAGL), comprised of a hexafluoroisopropyl (HFIP) carbamate warhead conjugated to the DHTz moiety. This sensitivity of the probe allowed it able to label endogenous MAGL in living PC3 cells, with an IC50 of 2.5 nm. Using both TIRF and conventional CLSM I was also able to spatiotemporally activate DHTz on the MAGL protein in live cells. This same methodology was used to image Bruton's tyrosine kinase (BTK), a protein which plays a crucial role in B cell development. Pfizer developed a BTK-DHTz probe using the covalent inhibitor Spebrutinib. I demonstrated its utility on a BTK-mCherry construct, achieving spatiotemporal labeling using CABL. CABL also allowed for an inverse-FRAP experiment to be performed, wherein a small subsection of a cell containing BTK-mCherry labeled with DHTz was spatiotemporally labeled with a TCO-fluorophore, and its diffusion away from the activation area was monitored. Attempts were made to develop reversible DHTz probes for bromodomain-containing protein 4 (BRD4) and microtubules, but unfortunately the attachment of DHTz to existing probes for these proteins inhibited their affinity, leading to poor signal localization. ☐ Lastly, in chapter 3 the discovery of the unique property of Tz to be reduced to its corresponding DHTz state is discussed. Conventional Tz probes that normally have display poor yields with TCO after extended incubation within the cellular milieu due to their reduction are able to have their reactivity rescued through application of the CABL. Furthermore, by pan-labeling the proteome of a living cell with a Tz-NHS ester, it was possible to observe how Tz is reduced by varying amounts within different locations of the cell. After pan-labeling cells with Tz and allowing for its reduction by the various cellular environments, the addition of a TCO-fluorophore revealed which areas of the cell retained Tz activity. By adding in a TCO-fluorophore of a different color and photooxidizing any reduced Tz using CABL, it was possible to visualize the heterogeneity of the redox state across the cell by CLSM on fixed and live cells, and by gel. This technique, dubbed CABL-R, was also used to examine the impact of inducing cellular redox stress with exogenous hydrogen peroxide, revealing that the areas of the cell such as the nucleus that normally maintain a reducing environment had their redox poise disturbed.
Description
Keywords
Bioorthogonal chemistry, Chemical biology, Tetrazine, Trans-cyclooctene