Biomolecular Perturbations in In-Cell Dynamic Nuclear Polarization Experiments

Abstract

In-cell DNP is a growing application of NMR to the study of biomolecular structure and function within intact cells. An important unresolved question for in-cell DNP spectroscopy is the integrity of cellular samples under the cryogenic conditions of DNP. Despite the rich literature around cryopreservation of cells in the fields of stem cell/embryonic cell therapeutics, cell line preservation and in cryo-EM applications, the effect of cryopreservation procedures on DNP parameters is unclear. In this report we investigate cell survival and apoptosis in the presence of cryopreserving agents and DNP radicals. We also assess the effects of these reagents on cellular enhancements. We show that the DNP radical AMUPol has no effect on membrane permeability and does not induce apoptosis. Furthermore, the standard aqueous glass forming reagent, comprised of 60/30/10 d8-glycerol/D2O/H2O (DNP juice), rapidly dehydrates cells and induces apoptosis prior to freezing, reducing structural integrity of the sample prior to DNP analysis. Preservation with d6-DMSO at 10% v/v provided similar DNP enhancements per √unit time compared to glycerol preservation with superior maintenance of cell size and membrane integrity prior to freezing. DMSO preservation also greatly enhanced post-thaw survival of cells slow-frozen at 1°C/min. We therefore demonstrate that in-cell DNP-NMR studies should be done with d6-DMSO as cryoprotectant and raise important considerations for the progression of in-cell DNP-NMR towards the goal of high quality structural studies.

In-Cell Quantification of Drugs by Magic-Angle Spinning Dynamic Nuclear Polarization NMR

“…Here, we used a protocol for sample preparation that has been widely used in the DNP community and also for biological samples. , The protocol is detrimental to the viability of the cells. , In the present study, however, we only require that the cells remain largely intact, without significant lysis, and we do not require the cells to be viable post-thawing. If cell viability post-thawing is required, this can be achieved for MAS DNP experiments using the recently introduced protocols ,,, that are otherwise perfectly compatible with the quantification approaches here.…”

“…More importantly, here, it requires the ability to quantitatively fully extract the compound of interest, which can be an issue when it is poorly soluble or engaged in strong interactions with, for example, a binding partner. Furthermore, we note that the most recent developments in sample preparation protocols for MAS DNP NMR allow not just for cells to remain intact but also for cells to remain viable following thawing, , which opens up the exciting possibility of following time courses of unmodified drugs using the NMR approach. The methodology introduced here is, of course, not intended to replace but rather to complement other quantification approaches currently used in biology.…”

“…In particular, the limit of detection can potentially be pushed further down with the detection of the more sensitive 19 F nucleus, especially given that it is present in many drugs and is almost absent in cells, obviating the problem of the cellular background. We expect that ongoing improvements in DNP instrumentation and the design of polarizing agents will further increase the sensitivity of the technique, , and the applications of the more recently introduced preparation protocols , will enhance the approach presented here. The present approach can also be affected if the drug is metabolized by the cells.…”

In-Cell NMR of Intact Mammalian Cells Preserved with the Cryoprotectants DMSO and Glycerol Have Similar DNP Performance

“…The most common cryoprotectant for cultured mammalian cells is dimethylsulfoxide (DMSO) at a concentration of 10% (v/v). Indeed, the handful of studies that examine preparations of mammalian cells using DNP NMR use DMSO ( Albert et al, 2018 ; Narasimhan et al, 2019 ; Schlagnitweit et al, 2019 ; Overall et al, 2020 ), although the sample composition—including the choice of cryoprotectant—and post-experiment cellular viability, have only very recently been considered ( Ghosh et al, 2020 ; Ghosh et al, 2021 ; Overall and Barnes, 2021 ). Given the widespread preference for 10% DMSO over 15% glycerol as the cryoprotectant for cellular cryopreservation, we sought to determine if cryoprotection using 10% DMSO could also support cellular viability throughout the DNP NMR experiments.…”

Solid-state NMR analysis of unlabeled fungal cell walls from Aspergillus and Candida species

“…The fungal cell walls, especially those of C. albicans , are substantially more mobile than the plant materials and are considered unfavorable for MAS-DNP. The success of natural-abundance DNP thus opens the frontier to interrogate other cellular systems with similar dynamical characteristics, such as microalgae, bacteria, and human and animal cells ( Takahashi, 2013 , Overall and Barnes, 2021 , Albert et al, 2018 , Romaniuk and Cegelski, 2015 , Poulhazan, 2021 , Poulhazan et al, 2018 , Arnold et al, 2018 , Ghosh et al, 2021 ), without isotopic enrichment.

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