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.
Highlights
In-cell NMR is the only technique that can provide structural, dynamic, and composition information of biomolecules in their native, intracellular context and at atomic resolution
In order to characterize the effect Dynamic nuclear polarization (DNP) preparation has on cell integrity and its relationship to DNP enhancements and spectral quality, we first assessed the cellular effects of DNP radicals and cryoprotecting reagents prior to cryogenic freezing
Cell viability was assessed using flow cytometry to measure forward light scatter, propidium iodide (PI) uptake and caspase-3 cleavage
Summary
In-cell NMR is the only technique that can provide structural, dynamic, and composition information of biomolecules in their native, intracellular context and at atomic resolution. Much of this work was pioneered by the work of Selenko and co-workers who demonstrated the transfer of high concentrations of uniformly labelled proteins into cells using electroporation and microinjection (Selenko et al, 2006) This circumvents some of the spectral complexity observed in in-cell NMR spectra. Subsequent studies used DNP-NMR to observe exogenously labelled ubiquitin electroporated into HeLa cells (Narasimhan et al, 2019), drug mediated expression of HIV particles (Overall et al, 2020) and antisense RNA drug complexes in HEK 293T cells (Schlagnitweit et al, 2019). Optimization of sample preparation and an understanding of the cellular effects of DNP preparation are lacking
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