Attionu Solomon K, Dill Rita, Summers Michael F, Case David A, Marchant Jan, Dayie Theodore K
Department of Chemistry and Biochemistry, University of Maryland, 8314 Paint Branch Dr, College Park, MD, 20742, USA.
Howard Hughes Medical Institute, University of Maryland, 1000 Hilltop Circle, Baltimore, MD, 21250, USA.
Chembiochem. 2025 Jun 16;26(12):e202500206. doi: 10.1002/cbic.202500206. Epub 2025 May 23.
RNAs regulate various cellular processes using malleable 3D structures, and understanding the factors that control RNA structure and dynamics is critical for understanding their mechanisms of action. To mitigate factors that have limited studies of large, functionally relevant RNAs by solution nuclear magnetic resonance (NMR) spectroscopy, we have extended a recently described H-enhanced, H-N correlation approach that used uniformly 15N-labeled guanosine triphosphate (GTP) by developing a chemoenzymatic labeling technology that grafts selectively labeled [9-N]-Guanine on to any labeled ribose to make [9-N]-GTP. The approach exploits advantageous NMR properties of the N9 nucleus which, when combined with extensive ribose deuteration and optimized NMR pulse sequences, affords sharp signals without complications that can arise using uniform [N]-guanine labeling. The utility of the approach for NMR signal assignment and dynamics analysis is demonstrated for three large RNAs (20-78 kDa) that play critical roles in viral replication. With this approach, NMR studies of RNAs comprising 200 nt or more should now be feasible.
RNA利用可塑的三维结构调控各种细胞过程，了解控制RNA结构和动力学的因素对于理解其作用机制至关重要。为减少因溶液核磁共振（NMR）光谱对大型功能相关RNA研究有限的因素，我们扩展了最近描述的H增强H-N相关方法，该方法使用均匀15N标记的鸟苷三磷酸（GTP），通过开发一种化学酶标记技术，将选择性标记的[9-N]-鸟嘌呤嫁接到任何标记的核糖上以制备[9-N]-GTP。该方法利用了N9原子核的有利NMR特性，当与广泛的核糖氘代和优化的NMR脉冲序列相结合时，可提供清晰的信号，而不会出现使用均匀[N]-鸟嘌呤标记可能产生的并发症。该方法在病毒复制中起关键作用的三种大型RNA（20-78 kDa）的NMR信号分配和动力学分析中的实用性得到了证明。通过这种方法，现在对包含200 nt或更多的RNA进行NMR研究应该是可行的。
