Although ∼98% of the human genomic output is transcribed as non-protein coding RNA, <2% of the protein data bank structures comprise RNA. This huge structural disparity stems from combined difficulties of crystallizing RNA for X-ray crystallography along with extensive chemical shift overlap and broadened linewidths associated with NMR of RNA. While half of the deposited RNA structures in the PDB were solved by NMR methods, the usefulness of NMR is still limited by the high cost of sample preparation and challenges of resonance assignment. Here we propose a novel strategy for resonance assignment that combines new strategic ¹³C labeling technologies with filter/edit type NOESY experiments to greatly reduce spectral complexity and crowding. This new strategy allowed us to assign important non-exchangeable resonances of proton and carbon (1′, 2′, 2, 5, 6 and 8) nuclei using only one sample and <24 h of NMR instrument time for a 27 nt model RNA. The method was further extended to assigning a 6 nt bulge from a 61 nt viral RNA element justifying its use for a wide range RNA chemical shift resonance assignment problems.

中文翻译：

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结合不对称¹³ C标记和同位素过滤/编辑NOESY：快速和逻辑RNA共振分配的新策略

尽管约98％的人类基因组输出被转录为非蛋白质编码的RNA，但不到2％的蛋白质数据库结构包含RNA。这种巨大的结构差异源于X射线晶体学中结晶RNA的困难，以及与RNA NMR相关的广泛的化学位移重叠和加宽的线宽。尽管PDB中一半的RNA结构已通过NMR方法解决，但NMR的有效性仍然受到样品制备成本高和共振分配挑战的限制。在这里，我们提出了一种新颖的共振分配策略，该策略结合了新的策略¹³带有过滤器/编辑型NOESY实验的C标记技术可大大降低频谱复杂性和拥挤程度。这一新策略使我们能够仅使用一个样品，并且对于27 nt模型RNA，使用NMR仪器时间<24小时，即可分配质子和碳（1'，2'，2、5、6和8）核的重要不可交换共振。该方法进一步扩展为从61 nt病毒RNA元件中分配6 nt凸起，证明其可用于广泛的RNA化学位移共振分配问题。