Selective Protein Hyperpolarization in Cell Lysates Using Targeted Dynamic Nuclear Polarization

Viennet, Thibault; Viegas, Aldino; Kuepper, Arne; Arens, Sabine; Gelev, Vladimir; Petrov, Ognyan; Grossmann, Tom N.; Heise, Henrike; Etzkorn, Manuel

doi:10.1002/ange.201603205

Cited by 35 publications

(26 citation statements)

References 30 publications

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“…Using the frozen‐cell DNP approach allows time to be stopped for a functional sample, removing acquisition time limitations due to potential cell death or further metabolization as well as size limitations of the molecule of interest. Together with on‐going improvements in intracellular localization of DNP radicals [30, 31] —TOTAPOL enhanced cell wall signals or nuclear localization of TotaFAM, for example—co‐localization with a transfected molecule can lead to localized enhancements of, for example, ASOs in different compartments in intact cells. We believe that this is just a first glimpse of the potential of this method.…”

Section: Figurementioning

confidence: 99%

Observing an Antisense Drug Complex in Intact Human Cells by in‐Cell NMR Spectroscopy

et al. 2019

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Gaining insight into the uptake, trafficking and target engagement of drugs in cells can enhance understanding of a drug's function and efficiency. However, there are currently no reliable methods for studying untagged biomolecules in macromolecular complexes in intact human cells. Here we have studied an antisense oligonucleotide (ASO) drug in HEK 293T and HeLa cells by NMR spectroscopy. Using a combination of transfection, cryoprotection and dynamic nuclear polarization (DNP), we were able to detect the drug directly in intact frozen cells. Activity of the drug was confirmed by quantitative reverse transcription polymerase chain reaction (qRT‐PCR). By applying DNP NMR to frozen cells, we overcame limitations both of solution‐state in‐cell NMR spectroscopy (e.g., size, stability and sensitivity) and of visualization techniques, in which (e.g., fluorescent) tagging of the ASO decreases its activity. The capability to detect an untagged, active drug, interacting in its natural environment, represents a first step towards studying molecular mechanisms in intact cells.

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Section: Figurementioning

confidence: 99%

Observing an Antisense Drug Complex in Intact Human Cells by in‐Cell NMR Spectroscopy

et al. 2019

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“…Cellular lysates and extracts have been used to approximate the cellular environment for DNP experiments 8,26 .We therefore tested the effect of cell lysis on the TOTAPOL reduction rate. To control for dilution effects in the lysate, we prepared whole-cell suspensions at the same concentration as the cell lysates.…”

Section: Totapol Reduction Is Slowed In Lysatesmentioning

confidence: 99%

Stability of Nitroxide Biradical TOTAPOL in Biological Samples

McCoy¹,

Rogawski

Stovicek

et al. 2019

Preprint

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We characterize chemical reduction of a nitroxide biradical, TOTAPOL, used in dynamic nuclear polarization (DNP) experiments, specifically probing the stability in whole-cell pellets and lysates, and present a few strategies to stabilize the biradicals for DNP studies. DNP solid-state NMR experiments use paramagnetic species such as nitroxide biradicals to dramatically increase NMR signals. Although there is considerable excitement about using nitroxide-based DNP for detecting the NMR spectra of proteins in whole cells, nitroxide radicals are reduced in minutes in bacterial cell pellets, which we confirm and quantify here. We show that addition of the covalent cysteine blocker Nethylmaleimide to whole cells significantly slows the rate of reduction, suggesting that cysteine thiol radicals are important to in vivo radical reduction. The use of cell lysates rather than whole cells also slows TOTAPOL reduction, which suggests a possible role for the periplasm and oxidative phosphorylation metabolites in radical degradation. Reduced TOTAPOL in lysates can also be efficiently reoxidized with potassium ferricyanide. These results point to a practical and robust set of strategies for DNP of cellular preparations.

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“…Alternatively, the design of targeted DNP polarization agents has been explored with selectivity achieved either through cysteine chemistry or protein-ligand interactions. [14][15][16][17][18][19][20] The applications of these strategies, however, are limited due to the reducing cellular environment or the requirement for a known synthetically accessible ligand of the target protein. We, therefore, sought to develop a general targeting strategy that can be applied to any protein of interest in a cysteine-independent manner, and that is compatible with DNP-enhanced NMR studies of proteins in the cellular interior.…”

mentioning

confidence: 99%

“…8). Using the background subtraction approach introduced by Viennet et al 18 , we estimate that the selective enhancement for conjugated ubiquitin is 5-fold greater than the enhancement of the lysate background. Our overall ubiquitin enhancement in this setting is ~10, which is on par with the enhancements we measured for similar concentrations of purified protein-TTz conjugates.…”

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confidence: 99%

Targetable Tetrazine-Based Dynamic Nuclear Polarization Agents for Biological Systems

Lim¹,

Ackermann²,

Debelouchina³

2019

Preprint

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Dynamic nuclear polarization (DNP) has shown great promise as a tool to enhance the nuclear magnetic resonance (NMR) signals of proteins in the cellular environment. As the sensitivity increases, the ability to select and efficiently polarize a specific macromolecule over the cellular background has become desirable. Here, we address this need and present a tetrazine-based DNP polarization agent that can be targeted selectively to proteins containing the unnatural amino acid (UAA) norbornene-lysine. The UAA can be introduced efficiently by genetic means in the cellular milieu. Our approach is bio-orthogonal and easily adaptable to any protein of interest. We illustrate the scope of our methodology and investigate the DNP polarization transfer mechanisms in several biological systems. Our results present the first molecular view of the complex polarization transfer pathways in targeted DNP and ultimately pave the way to selective DNP-enhanced NMR spectroscopy in both bacterial and mammalian cells.

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Selective Protein Hyperpolarization in Cell Lysates Using Targeted Dynamic Nuclear Polarization

Abstract: Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under http://dx.

Cited by 35 publications

References 30 publications

Observing an Antisense Drug Complex in Intact Human Cells by in‐Cell NMR Spectroscopy

Observing an Antisense Drug Complex in Intact Human Cells by in‐Cell NMR Spectroscopy

Stability of Nitroxide Biradical TOTAPOL in Biological Samples

Targetable Tetrazine-Based Dynamic Nuclear Polarization Agents for Biological Systems

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