Michnicka M J, Harper J W, King G C
W.M. Keck Center for Computational Biology, Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005.
Biochemistry. 1993 Jan 19;32(2):395-400. doi: 10.1021/bi00053a002.
The introduction of isotopically enriched nucleotides into NMR quantities of a synthetic 29-mer RNA derived from the HIV-1 TAR element is described. RNA enriched in 13C and/or 15N is produced by a procedure which involves isolation of whole cellular RNA from Escherichia coli, nucleolysis, separation of mononucleotides, chemical or enzymatic pyrophosphorylation, and in vitro transcription by T7 RNA polymerase. Spectral characteristics of each residue type are examined in isolation. 13C chemical shifts provide an alternative method to determine ribose puckers for larger RNAs. Nonprotonated sites such as purine N7 groups can now be monitored through the use of multiple-bond 1H-15N coupling. When applied conservatively, coordinate analysis of chemical shift values should prove valuable for NMR studies of RNA structure and recognition. 1H, 13C, and 15N chemical shift data suggest that TAR residue A35 has an unusual local environment, consistent with extrusion of its base from the terminal loop.
本文描述了将同位素富集的核苷酸引入源自HIV-1 TAR元件的合成29聚体RNA的核磁共振（NMR）量中的方法。富含13C和/或15N的RNA是通过以下步骤产生的：从大肠杆菌中分离全细胞RNA、核酸分解、单核苷酸分离、化学或酶促焦磷酸化以及T7 RNA聚合酶的体外转录。分别检查每种残基类型的光谱特征。13C化学位移为确定较大RNA的核糖构象提供了另一种方法。现在可以通过使用多键1H-15N耦合来监测非质子化位点，如嘌呤N7基团。如果保守应用，化学位移值的坐标分析对于RNA结构和识别的NMR研究应该是有价值的。1H、13C和15N化学位移数据表明，TAR残基A35具有异常的局部环境，与其碱基从末端环挤出一致。
