Biophysics Program, Department of Chemistry, Biomedical Engineering, and Macromolecular Science and Engineering, Michigan Neuroscience Institute, University of Michigan, Ann Arbor, MI 48109, USA.
National Magnetic Resonance Facility at Madison, University of Wisconsin-Madison, Madison, WI 53706, USA.
Biomolecules. 2022 Nov 3;12(11):1628. doi: 10.3390/biom12111628.
Residual dipolar couplings (RDCs) are increasingly used for high-throughput NMR-based structural studies and to provide long-range angular constraints to validate and refine structures of various molecules determined by X-ray crystallography and NMR spectroscopy. RDCs of a given molecule can be measured in an anisotropic environment that aligns in an external magnetic field. Here, we demonstrate the first application of polymer-based nanodiscs for the measurement of RDCs from nucleic acids. Polymer-based nanodiscs prepared using negatively charged SMA-EA polymer and zwitterionic DMPC lipids were characterized by size-exclusion chromatography, H NMR, dynamic light-scattering, and H NMR. The magnetically aligned polymer-nanodiscs were used as an alignment medium to measure RDCs from a C/N-labeled fluoride riboswitch aptamer using 2D ARTSY-HSQC NMR experiments. The results showed that the alignment of nanodiscs is stable for nucleic acids and nanodisc-induced RDCs fit well with the previously determined solution structure of the riboswitch. These results demonstrate that SMA-EA-based lipid-nanodiscs can be used as a stable alignment medium for high-resolution structural and dynamical studies of nucleic acids, and they can also be applicable to study various other biomolecules and small molecules in general.
残基偶极耦合（RDC）越来越多地用于基于 NMR 的高通量结构研究，并提供远程角度约束，以验证和细化通过 X 射线晶体学和 NMR 光谱学确定的各种分子的结构。在外部磁场中排列的各向异性环境中可以测量给定分子的 RDC。在这里，我们展示了聚合物纳米盘在测量核酸 RDC 方面的首次应用。使用带负电荷的 SMA-EA 聚合物和两性离子 DMPC 脂质制备的聚合物纳米盘通过尺寸排阻色谱法、H NMR、动态光散射和 H NMR 进行了表征。将磁定向聚合物纳米盘用作对齐介质，使用 2D ARTSY-HSQC NMR 实验测量 C/N 标记的氟化物核糖开关适体的 RDC。结果表明，纳米盘的对齐对于核酸是稳定的，并且纳米盘诱导的 RDC 与先前确定的核糖开关溶液结构很好地吻合。这些结果表明，基于 SMA-EA 的脂质纳米盘可作为核酸高分辨率结构和动力学研究的稳定对齐介质，并且通常也可适用于研究各种其他生物分子和小分子。
