Department of Cell Biology and Molecular Genetics, University of Maryland, 4062 Campus Dr., College Park, MD, 20742, USA.
Center for Biomolecular Structure & Organization, Department of Chemistry & Biochemistry, University of Maryland, 8314 Paint Branch Dr., College Park, MD, 20782, USA.
Chemistry. 2018 Apr 11;24(21):5462-5468. doi: 10.1002/chem.201705948. Epub 2018 Mar 25.
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 C 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.
RNA 分子的构象动力学在调节其生物功能方面起着至关重要的作用。测量 RNA 的动态行为可以深入了解与 RNA 相互作用的结合伙伴或细胞刺激物的相关信息。然而，由于严重的光谱重叠、同核 C 标量偶合和谱线增宽，使用溶液状态 NMR 对大于 70 个核苷酸（nt=核苷酸）的大型 RNA 分子进行此类测量非常困难。本文引入了固相合成、定点同位素标记的亚磷酰胺和酶连接的策略组合。这种方法可以将同位素探针精确地插入到 96 个核苷酸的 CCR5 RNA 片段中。通过使用 Carr-Purcell-Meiboom-Gill（CPMG）弛豫分散（RD）实验进行功能动力学的精确测量，可以提取该 RNA 的交换速率和群体。对 RNA/ microRNA-1224 复合物的 NMR 化学位移扰动分析表明，假结的 A90-C1'表现出与激发态中观察到的相似的化学位移变化。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。
