Stability of the nitroxide biradical AMUPol in intact and lysed mammalian cells

Dynamic Nuclear Polarization (DNP) enhanced solid state NMR increases experimental sensitivity, potentially enabling detection of biomolecules at their physiological concentrations. The sensitivity of DNP experiments is due to the transfer of polarization from electron spins of free radicals to the nuclear spins of interest. Here, we investigate the reduction of AMUPol in both lysed and intact HEK293 cells. We find that nitroxide radicals are reduced with first order reduction kinetics by cell lysates at a rate of ti 12% of the added nitroxide radical concentration per hour. We also found that electroporation delivered a consistent amount of AMUPol to intact cells and that nitroxide radicals are reduced just slightly more rapidly (-15% per hour) by intact cells than by cell lysates. The two nitroxide radicals of AMUPol are reduced independently and this leads to considerable accumulation of the DNP-silent monoradical form of AMUPol, particularly in preparations of intact cells where nearly half of the AMUPol is already reduced to the DNP silent monoradical form at the earliest experimental time points. This confirms that the loss of the DNP-active biradical form of AMUPol is faster than the nitroxide reduction rate. Finally, we investigate the effect of adding N-ethyl maleimide, a well-known inhibitor of thiol (-SH) group-based reduction of nitroxide biradicals in cells, on AMUPol reduction, cellular viability, and DNP performance. Although pre-treatment of cells with NEM effectively inhibited the reduction of AMUPol, exposure to NEM compromised cellular viability and, surprisingly, did not improve DNP performance. Collectively, these results indicate that, currently, the most effective strategy to obtain high DNP enhancements for DNP-assisted in-cell NMR is to minimize room temperature contact times with cellular constituents and suggest that the development of bio-resistant polarization agents for DNP could considerably increase the sensitivity of DNP-assisted in-cell NMR experiments. (c) 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Automated summary

Dynamic nuclear polarization (DNP) enhanced solid state NMR increases sensitivity, allowing detection of biomolecules at physiological concentrations. Reduction of AMUPol in lysed and intact HEK293 cells was investigated, showing first-order reduction kinetics by cell lysates and slightly faster reduction by intact cells. Independent reduction of the two nitroxide radicals of AMUPol leads to accumulation of the DNP-silent monoradical form. The addition of N-ethyl maleimide (NEM) inhibits AMUPol reduction but compromises cellular viability and does not improve DNP performance. Minimizing room temperature contact times with cellular constituents is the most effective strategy for achieving high DNP enhancements in in-cell NMR experiments.