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Dynamic Nuclear Polarization Nuclear Magnetic Resonance in Human Cells Using Fluorescent Polarizing Agents
Biochemistry ( IF 2.9 ) Pub Date : 2018-06-20 00:00:00 , DOI: 10.1021/acs.biochem.8b00257 Brice J. Albert 1 , Chukun Gao 1 , Erika L. Sesti 1 , Edward P. Saliba 1 , Nicholas Alaniva 1 , Faith J. Scott 1 , Snorri Th. Sigurdsson 2 , Alexander B. Barnes 1
Biochemistry ( IF 2.9 ) Pub Date : 2018-06-20 00:00:00 , DOI: 10.1021/acs.biochem.8b00257 Brice J. Albert 1 , Chukun Gao 1 , Erika L. Sesti 1 , Edward P. Saliba 1 , Nicholas Alaniva 1 , Faith J. Scott 1 , Snorri Th. Sigurdsson 2 , Alexander B. Barnes 1
Affiliation
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Solid state nuclear magnetic resonance (NMR) enables atomic-resolution characterization of the molecular structure and dynamics within complex heterogeneous samples, but it is typically insensitive. Dynamic nuclear polarization (DNP) increases the NMR signal intensity by orders of magnitude and can be performed in combination with magic angle spinning (MAS) for sensitive, high-resolution spectroscopy. Here we report MAS DNP experiments, for the first time, within intact human cells with >40-fold DNP enhancement and a sample temperature of <6 K. In addition to cryogenic MAS results at <6 K, we also show in-cell DNP enhancements of 57-fold at 90 K. In-cell DNP is demonstrated using biradicals and sterically shielded monoradicals as polarizing agents. A novel trimodal polarizing agent is introduced for DNP, which contains a nitroxide biradical, a targeting peptide for cell penetration, and a fluorophore for subcellular localization with confocal microscopy. The fluorescent polarizing agent provides in-cell DNP enhancements of 63-fold at a concentration of 2.7 mM. These experiments pave the way for structural characterization of biomolecules in an endogenous cellular context.
中文翻译：
使用荧光偏振剂的人类细胞中的动态核极化核磁共振
固态核磁共振（NMR）能够对复杂的异质样品中的分子结构和动力学进行原子分辨率表征，但通常不敏感。动态核极化（DNP）将NMR信号强度提高了几个数量级，可以与灵敏角旋转（MAS）结合使用，以实现灵敏的高分辨率光谱。在这里，我们首次报告了具有完整DNP增强> 40倍且样品温度<6 K的完整人类细胞内的MAS DNP实验。除了在<6 K时的低温MAS结果，我们还显示了细胞内DNP在90 K时增强了57倍。使用双基和空间屏蔽单基作为极化剂证明了细胞内DNP。引入了用于DNP的新型三峰偏振剂，它包含一氧化氮双自由基，用于细胞渗透的靶向肽和用于通过共聚焦显微镜进行亚细胞定位的荧光团。荧光偏振剂在2.7 mM的浓度下可将细胞内DNP增强63倍。这些实验为在内源性细胞环境中生物分子的结构表征铺平了道路。
更新日期：2018-06-20
中文翻译：
使用荧光偏振剂的人类细胞中的动态核极化核磁共振
固态核磁共振（NMR）能够对复杂的异质样品中的分子结构和动力学进行原子分辨率表征，但通常不敏感。动态核极化（DNP）将NMR信号强度提高了几个数量级，可以与灵敏角旋转（MAS）结合使用，以实现灵敏的高分辨率光谱。在这里，我们首次报告了具有完整DNP增强> 40倍且样品温度<6 K的完整人类细胞内的MAS DNP实验。除了在<6 K时的低温MAS结果，我们还显示了细胞内DNP在90 K时增强了57倍。使用双基和空间屏蔽单基作为极化剂证明了细胞内DNP。引入了用于DNP的新型三峰偏振剂，它包含一氧化氮双自由基，用于细胞渗透的靶向肽和用于通过共聚焦显微镜进行亚细胞定位的荧光团。荧光偏振剂在2.7 mM的浓度下可将细胞内DNP增强63倍。这些实验为在内源性细胞环境中生物分子的结构表征铺平了道路。
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