PREPARATION OF ISOTOPICALLY LABELED RIBONUCLEOTIDES FOR MULTIDIMENSIONAL NMR-SPECTROSCOPY OF RNA
PREPARATION OF ISOTOPICALLY LABELED RIBONUCLEOTIDES FOR MULTIDIMENSIONAL NMR-SPECTROSCOPY OF RNA
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DOI：
10.1093/nar/20.17.4515
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发表时间：
1992-09-11
影响因子：
14.9
通讯作者：
WILLIAMSON, JR
中科院分区：
文献类型：
--
作者：
BATEY, RT;INADA, M;WILLIAMSON, JR
描述了大规模制备均匀同位素标记的核糖核苷酸和 RNA 的一般方法。细菌在同位素生长培养基上生长，收获它们的核酸并将其降解为单核苷酸。它们被酶促转化为核糖核苷三磷酸，用于体外转录反应以制备用于 NMR 研究的 RNA。对于 N-15 标记，大肠杆菌在 N-15-硫酸铵上生长，而对于 C-13 标记，嗜甲基菌在 C-13-甲醇上生长，这比 C-13-葡萄糖更经济。为了证明该方法的可行性和实用性，使用统一 C-13 标记的核糖核苷酸合成 31 核苷酸的 HIV TAR RNA，并通过 3D-NMR 进行分析。这种方法应该在通过 NMR 进行的 RNA 结构分析中得到广泛应用。
A general method for large scale preparation of uniformly isotopically labeled ribonucleotides and RNAs is described. Bacteria are grown on isotopic growth medium, and their nucleic acids are harvested and degraded to mononucleotides. These are enzymatically converted into ribonucleoside triphosphates, which are used in transcription reactions in vitro to prepare RNAs for NMR studies. For N-15-labeling, E.coli is grown on N-15-ammonium sulfate, whereas for C-13-labeling, Methylophilus methylotrophus is grown on C-13-methanol, which is more economical than C-13-glucose. To demonstrate the feasibility and utility of this method, uniformly C-13-labeled ribonucleotides were used to synthesize a 31 nucleotide HIV TAR RNA that was analyzed by 3D-NMR. This method should find widespread use in the structural analysis of RNA by NMR.