Krähenbühl Barbara, Lukavsky Peter, Wider Gerhard
Institute of Molecular Biology and Biophysics, ETH Zurich, 8093, Zurich, Switzerland.
J Biomol NMR. 2014 Aug;59(4):231-40. doi: 10.1007/s10858-014-9841-3. Epub 2014 Jun 5.
A procedure is presented for automated sequence-specific assignment of NMR resonances of uniformly [(13)C, (15)N]-labeled RNA. The method is based on a suite of four through-bond and two through-space high-dimensional automated projection spectroscopy (APSY) experiments. The approach is exemplified with a 0.3 mM sample of an RNA stem-loop with 48 nucleotides, K10, which is responsible for dynein-mediated localization of Drosophila fs(1)K10 mRNA transcripts. The automated analysis of the APSY data led to highly accurate and precise 3- to 4-dimensional peak lists. They provided a reliable basis for the subsequent sequence-specific resonance assignment with the algorithm FLYA and resulted in the fully automated resonance assignment of more than 80 % of the resonances of the (13)C-(1)H moieties at the 1', 2', 5, 6, and 8 positions in the nucleotides. The procedure was robust with respect to numerous impurity peaks, low concentration of this for NMR comparably large RNA, and structural features such as a loop, single-nucleotide bulges and a non-Watson-Crick wobble base pairs. Currently, there is no precise chemical shift statistics (as used by FLYA) for RNA regions which deviate from the regular A-form helical structure. Reliable and precise peak lists are thus required for automated sequence-specific assignment, as provided by APSY.
本文介绍了一种用于对均匀[(13)C, (15)N]标记的RNA的核磁共振（NMR）共振进行自动序列特异性归属的方法。该方法基于一套四个通过键合和两个通过空间的高维自动投影光谱（APSY）实验。以一个含有48个核苷酸的RNA茎环K10的0.3 mM样品为例进行了说明，该茎环负责动力蛋白介导的果蝇fs(1)K10 mRNA转录本的定位。对APSY数据的自动分析产生了高度准确和精确的3至4维峰列表。它们为随后使用FLYA算法进行序列特异性共振归属提供了可靠的基础，并实现了对核苷酸中1'、2'、5、6和8位上(13)C-(1)H基团80%以上共振的全自动归属。该方法对于众多杂质峰、这种相对较大的用于NMR的RNA的低浓度以及诸如环、单核苷酸凸起和非沃森-克里克摆动碱基对等结构特征具有稳健性。目前，对于偏离规则A-型螺旋结构的RNA区域，尚无精确的化学位移统计数据（如FLYA所使用的）。因此，如APSY所提供的那样，可靠且精确的峰列表对于自动序列特异性归属是必需的。
