Preparation and characterization of a uniformly 2 H/ 15 N-labeled RNA oligonucleotide for NMR studies.
Preparation and characterization of a uniformly 2 H/ 15 N-labeled RNA oligonucleotide for NMR studies.
复制标题
用于 NMR 研究的均匀 2 H/ 15 N 标记 RNA 寡核苷酸的制备和表征。
DOI：
10.1093/nar/25.7.1390
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发表时间：
1997
影响因子：
14.9
通讯作者：
DeJong,E
中科院分区：
文献类型：
--
作者：
Nikonowicz,EP;Michnicka,M;Kalurachchi,K;DeJong,E
用酶法制备了一种含有与核糖体蛋白S8结合位点的RNA寡核苷酸，在所有非交换位点上进行了均匀的15 N同位素富集和均匀的氘富集。含有44个核苷酸的RNA结合位点在溶液中形成发夹，并且需要Mg 2+进行适当折叠。比较了[2 H15 N]富集的RNA分子和相应的完全[2 H15 N]富集的RNA发夹的可交换质子的纵向磁化恢复率。发现1H-1H偶极弛豫对可交换质子纵向磁化强度的恢复有重要贡献。可交换质子共振谱线宽度受氘代的影响较小，表明与H2O的化学交换仍然是横向磁化弛豫的主导机制。然而，发现该RNA发夹的氘化相对于完全质子化的发夹增强了基于NOE的实验的灵敏度，并简化了2D NMR光谱。增加的信噪比促进了胞苷氨基共振和几个嘌呤核苷酸氨基共振的分配，并允许识别在完全质子化的RNA发夹的光谱中无法观察到的NOE交叉。
An RNA oligonucleotide that contains the binding site forEscherichiaco//ribosomal protein S8 was prepared with uniform15N isotopic enrichment and uniform deuterium enrichment at all non-exchangeable sites using enzymatic methods. The RNA binding site which contains 44 nt forms a hairpin in solution and requires Mg2+for proper folding. The longitudinal magnetization recovery rates of the exchangeable protons were compared for the [2H15N]-enriched RNA molecule and for the corresponding fully [2H15N]-enriched RNA hairpin. It was found that1H-1H dipolar relaxation significantly contributes to the recovery of exchangeable proton longitudinal magnetization. The exchangeable proton resonance line widths were less affected by deuteration indicating that chemical exchange with H2O remains the dominant mechanism of transverse magnetization relaxation. Nevertheless deuteration of this RNA hairpin was found to enhance the sensitivity of NOE-based experiments relative to the fully protonated hairpin and to simplify 2D NMR spectra. The increased signal-to-noise ratio facilitated the assignment of the cytidine amino resonances and several of the purine nucleotide amino resonances and permitted the identification of NOE crosspeaks that could not be observed in spectra of the fully protonated RNA hairpin.