Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance (BMRZ), Goethe-Universität Frankfurt, 60438 Frankfurt, Germany.
Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance (BMRZ), Goethe-Universität Frankfurt, 60438 Frankfurt, Germany
RNA. 2024 Jun 17;30(7):779-794. doi: 10.1261/rna.079902.123.
The stem-loop 2 motif (s2m) in SARS-CoV-2 (SCoV-2) is located in the 3'-UTR. Although s2m has been reported to display characteristics of a mobile genomic element that might lead to an evolutionary advantage, its function has remained unknown. The secondary structure of the original SCoV-2 RNA sequence (Wuhan-Hu-1) was determined by NMR in late 2020, delineating the base-pairing pattern and revealing substantial differences in secondary structure compared to SARS-CoV-1 (SCoV-1). The existence of a single G29742-A29756 mismatch in the upper stem of s2m leads to its destabilization and impedes a complete NMR analysis. With Delta, a variant of concern has evolved with one mutation compared to the original sequence that replaces G29742 by U29742. We show here that this mutation results in a more defined structure at ambient temperature accompanied by a rise in melting temperature. Consequently, we were able to identify >90% of the relevant NMR resonances using a combination of selective RNA labeling and filtered 2D NOESY as well as 4D NMR experiments. We present a comprehensive NMR analysis of the secondary structure, (sub)nanosecond dynamics, and ribose conformation of s2m Delta based on heteronuclear C NOE and measurements and ribose carbon chemical shift-derived canonical coordinates. We further show that the G29742U mutation in Delta has no influence on the druggability of s2m compared to the Wuhan-Hu-1 sequence. With the assignment at hand, we identify the flexible regions of s2m as the primary site for small molecule binding.
SARS-CoV-2（SCoV-2）的茎环 2 基序（s2m）位于 3'-UTR 中。尽管已经报道 s2m 具有移动基因组元件的特征，可能导致进化优势，但它的功能仍然未知。2020 年底，通过 NMR 确定了原始 SCoV-2 RNA 序列（武汉-Hu-1）的二级结构，描绘了碱基配对模式，并揭示了与 SARS-CoV-1（SCoV-1）相比，二级结构存在显著差异。s2m 上茎中的单个 G29742-A29756 不匹配导致其失稳，并阻碍了完整的 NMR 分析。与 Delta 变体相比，原始序列发生了一个突变，用 U29742 替换了 G29742。我们在这里表明，这种突变导致在环境温度下形成更明确的结构，同时熔点升高。因此，我们能够使用选择性 RNA 标记和过滤 2D NOESY 以及 4D NMR 实验的组合，鉴定出 >90%的相关 NMR 共振。我们基于异核 C NOE 和 测量以及核糖碳化学位移衍生的规范坐标，对 s2m Delta 的二级结构、（亚）纳秒动力学和核糖构象进行了全面的 NMR 分析。我们进一步表明，与武汉-Hu-1 序列相比，Delta 中的 G29742U 突变对 s2m 的成药性没有影响。有了这个分配，我们确定了 s2m 的柔性区域是小分子结合的主要部位。
