High-resolution NMR study of a synthetic DNA-RNA hybrid dodecamer containing the consensus pribnow promoter sequence: d(CGTTATAATGCG).r(CGCAUUAUAACG).
High-resolution NMR study of a synthetic DNA-RNA hybrid dodecamer containing the consensus pribnow promoter sequence: d(CGTTATAATGCG).r(CGCAUUAUAACG).
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含有共有 pribnow 启动子序列的合成 DNA-RNA 杂合十二聚体的高分辨率 NMR 研究：d(CGTTATAATGCG).r(CGCAUUAUAACG)。
DOI：
10.1021/bi00432a014
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发表时间：
1989
期刊：
影响因子：
2.9
通讯作者：
Reid,B
中科院分区：
文献类型：
--
作者：
Chou,SH;Flynn,P;Reid,B
材料和方法使用在 2'-位置用 TBDMS 保护的 5'-DMT 3'-(iV，iV-二异丙基甲氧基亚磷酰胺)快速有效地合成固相 RNA 的程序已在前面的论文中进行了描述。在自动DNA合成仪（Applied Biosystems Model 380B）上分别进行10微摩尔RNA和DNA合成。 d (CGTTATAATGCG) 和 r (CGCAUUAUAACG) 产物通过分析 TLC 纯化，如前所述 [参见前述论文 (Chou 等人，1989)]。将等量（约 10 mg）的每条链混合在 50 mL 10 mM 磷酸盐缓冲液中，加热至 80°C，缓慢冷却至室温，然后在羟基磷灰石柱上用磷酸盐梯度进行色谱分离，以去除单链材料，如前文所述。核磁共振波谱。本研究中使用的所有 NMR 实验均在温度为 31°C 的 Bruker WM-500 NMR 波谱仪上进行。可交换质子谱是通过 Redfield 溶剂抑制方案获得的，载波频率设置为大约 12 ppm，总脉冲长度为 275 µ&。 NOE 差异光谱是通过在所需共振位置处施加射频 (rf) 辐射对 16 次扫描进行平均，并从射频偏共振的等量扫描中减去该总和来收集的；总共累积了 3200 次扫描。从 t2 中的 1024 个复杂点和 400 个 tx 点收集了绝对幅度 COSY 数据。混合时间为 100 和 300 ms 的 NOESY 光谱是在相敏模式下（States 等，1982）从 1024 个复数 t2 点和 400 对实部和虚部 t {实验中获取的。
Materials and MethodsThe procedure for rapid and efficient solid-phase RNA synthesis using 5'-DMT 3'-(iV, iV-diisopropylmethoxy-phosphoramidites) protected with TBDMS at the 2'-position was described in the preceding paper. Ten-micromole RNA and DNA syntheses were carried out separately on an automated DNA synthesizer (Applied Biosystems Model 380B). The d (CGTTATAATGCG) and r (CGCAUUAUAACG) products were purified by analytical TLC, as described pre-viously [see preceding paper (Chou et al., 1989)]. Equal amounts (about 10 mg) of each strand were combined in 50 mL of 10 mM phosphate buffer, heated to 80 C, slowly cooled to room temperature, and then chromatographed on a hy-droxyapatite column with a phosphate gradient to remove single-stranded material, as described in the preceding paper. NMR Spectroscopy. All NMR experiments used in this study were performed on a Bruker WM-500 NMR spec-trometer at a temperature of 31 C. Exchangeable proton spectra were obtained with a Redfield solvent-suppression scheme with the carrier frequency set at approximately 12 ppm and a total pulse length of 275 µ&. NOE difference spectra were collected by coaveraging 16 scans with applied radio-frequency (rf) irradiation at a desired resonance position and subtracting that sum from an equal number of scans with the rf off-resonance; a total of 3200 scans were accumulated.Absolute-magnitude COSY data were collected from 1024 complex points in t2 and 400 tx points. NOESY spectra with mixing times of 100 and 300 ms were acquired in the phase-sensitive mode (States et al., 1982), from 1024 complex t2 points and 400 pairs of real and imaginary t {experiments.