Conformational dynamics of RNA molecules play a critical role in governing their biological functions. Measurements of RNA dynamic behavior sheds important light on sites that interact with their binding partners or cellular stimulators. However, such measurements using solution‐state NMR are difficult for large RNA molecules (>70 nt; nt=nucleotides) owing to severe spectral overlap, homonuclear ¹³C scalar couplings, and line broadening. Herein, a strategic combination of solid‐phase synthesis, site‐specific isotopic labeled phosphoramidites, and enzymatic ligation is introduced. This approach allowed the position‐specific insertion of isotopic probes into a 96 nt CCR5 RNA fragment. Accurate measurements of functional dynamics using the Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion (RD) experiments enabled extraction of the exchange rates and populations of this RNA. NMR chemical shift perturbation analysis of the RNA/microRNA‐1224 complex indicated that A90‐C1′ of the pseudoknot exhibits similar changes in chemical shift observed in the excited state. This work demonstrates the general applicability of a NMR‐labeling strategy to probe functional RNA structural dynamics.

中文翻译：

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CCR5 RNA假结：残基和位点特异性标记将内部运动与microRNA结合相关

RNA分子的构象动力学在控制其生物学功能中起着至关重要的作用。RNA动态行为的测量为与其结合伴侣或细胞刺激物相互作用的位点提供了重要的启示。但是，由于严重的光谱重叠，同核¹³ C标量耦合和谱线加宽，使用溶液状态NMR进行的此类测量对于大型RNA分子（> 70 nt； nt =核苷酸）而言是困难的。本文介绍了固相合成，位点特异性同位素标记的亚磷酰胺和酶促连接的战略组合。这种方法允许将同位素探针特定位置插入96 nt CCR中5 RNA片段。使用Carr–Purcell–Meiboom–Gill（CPMG）弛豫分散（RD）实验对功能动力学进行准确测量，可以提取该RNA的交换速率和种群。对RNA / microRNA-1224复合物的NMR化学位移扰动分析表明，假结的A90-C1'在激发态下观察到的化学位移具有相似的变化。这项工作证明了NMR标记策略在探测功能性RNA结构动力学方面的普遍适用性。