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Cryogenic magic angle spinning (MAS) is a standard technique utilized for dynamic nuclear polarization (DNP) in solid-state nuclear magnetic resonance (NMR). Here we describe the optimization and implementation of a stator for cryogenic MAS with 9.5 mm diameter spherical rotors, allowing for DNP experiments on large sample volumes. Designs of the stator and rotor for cryogenic MAS build on recent advancements of MAS spheres and take a step further to incorporate sample insert and eject and a temperature-independent spinning stability of ±1 Hz. At a field of 7 T and spinning at 2.0 kHz with a sample temperature of 105–107 K, DNP enhancements of 256 and 200 were observed for 124 and 223 µ L sample volumes, respectively, each consisting of 4 M 13 C, 15 N-labeled urea and 20 mM AMUPol in a glycerol–water glassy matrix. [ABSTRACT FROM AUTHOR]

Cryogenic magic-angle spinning (MAS) is a standard technique utilized for Dynamic Nuclear Polarization (DNP) in solid state nuclear magnetic resonance (NMR). Here we describe the optimization and implementation of a stator for cryogenic MAS with spherical rotors, allowing for DNP experiments on large sample volumes. Designs of the stator and rotor for cryogenic MAS build on recent advancements of MAS spheres, and take a step further to incorporate sample-insert/eject and temperature-independent spinning stability of +/- 1 Hz. At a field of 7 T and spinning at 2.0 kHz with a sample temperature of 105–107 K, DNP enhancements of 256 and 200 were observed for 124 μL and 223 μL sample volumes, respectively, each consisting of 4 M 13C, 15N-labelled urea and 20 mM AMUPol in a glycerol-water glassy matrix. [ABSTRACT FROM AUTHOR]

The use of spherical rotors for magic angle spinning offers a number of advantages, including improved sample exchange, efficient microwave coupling for dynamic nuclear polarization nuclear magnetic resonance (NMR) experiments, and, most significantly, high frequency and stable spinning with minimal risk of rotor crash. Here we demonstrate the simple retrofitting of a commercial NMR probe with MAS spheres for solid-state NMR. We analyze a series of turbine groove geometries to investigate the importance of the rotor surface for spinning performance. Of note, rotors lacking any surface modification spin rapidly and stably even without feedback control. The high stability of a spherical rotor about the magic angle is shown to be dependent on its inertia tensor rather than the presence of turbine grooves. [ABSTRACT FROM AUTHOR]

We demonstrate for the first time in-cell dynamic nuclear polarization (DNP) in conjunction with flow cytometry sorting to address the cellular heterogeneity of in-cell samples. Utilizing a green fluorescent protein (GFP) reporter of HIV reactivation, we correlate increased 15N resonance intensity with cytokine-driven HIV reactivation in a human cell line model of HIV latency. As few as 10% GFP+ cells could be detected by DNP nuclear magnetic resonance (NMR). The inclusion of flow cytometric sorting of GFP+ cells prior to analysis by DNP-NMR further boosted signal detection through increased cellular homogeneity with respect to GFP expression. As few as 3.6 million 15N-labeled GFP+ cells could be readily detected with DNP-NMR. Importantly, cell sorting allowed for the comparison of cytokine-treated GFP+ and GFP− cells in a batch-consistent way. This provides an avenue for normalizing NMR spectral contributions from background cellular processes following treatment with cellular modulators. We also demonstrate the remarkable stability of AMUPol (a nitroxide biradical) in Jurkat T cells and achieved in-cell enhancements of 46 with 10 mM AMUPol, providing an excellent model system for further in-cell DNP-NMR studies. This represents an important contribution to improving in-cell methods for the study of endogenously expressed proteins by DNP-NMR. [ABSTRACT FROM AUTHOR]