Cryo-EM and antisense targeting of the 28-kDa frameshift stimulation element from the SARS-CoV-2 RNA genome,Nature Structural & Molecular Biology

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Cryo-EM and antisense targeting of the 28-kDa frameshift stimulation element from the SARS-CoV-2 RNA genome
Nature Structural & Molecular Biology ( IF 12.5 ) Pub Date : 2021-08-23 , DOI: 10.1038/s41594-021-00653-y
Kaiming Zhang _{1,

2} , Ivan N Zheludev ₃ , Rachel J Hagey ₄ , Raphael Haslecker ₅ , Yixuan J Hou ₆ , Rachael Kretsch ₇ , Grigore D Pintilie ₁ , Ramya Rangan ₇ , Wipapat Kladwang ₃ , Shanshan Li _{1,

2} , Marie Teng-Pei Wu ₅ , Edward A Pham ₄ , Claire Bernardin-Souibgui ₄ , Ralph S Baric _{6,

8} , Timothy P Sheahan ₆ , Victoria D'Souza ₅ , Jeffrey S Glenn _{4,

9} , Wah Chiu _{1,

7,

10} , Rhiju Das _{3,

7,

11}

Affiliation

Departments of Bioengineering, James H. Clark Center, Stanford University, Stanford, CA, USA.
MOE Key Laboratory for Membraneless Organelles and Cellular Dynamics, Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, China.
Department of Biochemistry Stanford University, Stanford, CA, USA.
Departments of Medicine (Division of Gastroenterology and Hepatology) and Microbiology & Immunology, Stanford School of Medicine, Stanford, CA, USA.
Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA.
Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
Biophysics Program, Stanford University, Stanford, CA, USA.
Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
Palo Alto Veterans Administration, Palo Alto, CA, USA.
CryoEM and Bioimaging Division, Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, CA, USA.
Department of Physics, Stanford University, Stanford, CA, USA.

Drug discovery campaigns against COVID-19 are beginning to target the SARS-CoV-2 RNA genome. The highly conserved frameshift stimulation element (FSE), required for balanced expression of viral proteins, is a particularly attractive SARS-CoV-2 RNA target. Here we present a 6.9 Å resolution cryo-EM structure of the FSE (88 nucleotides, ~28 kDa), validated through an RNA nanostructure tagging method. The tertiary structure presents a topologically complex fold in which the 5′ end is threaded through a ring formed inside a three-stem pseudoknot. Guided by this structure, we develop antisense oligonucleotides that impair FSE function in frameshifting assays and knock down SARS-CoV-2 virus replication in A549-ACE2 cells at 100 nM concentration.

中文翻译：

SARS-CoV-2 RNA 基因组中 28 kDa 移码刺激元件的冷冻电镜和反义靶向

针对 COVID-19 的药物发现活动开始针对 SARS-CoV-2 RNA 基因组。病毒蛋白平衡表达所需的高度保守的移码刺激元件 (FSE) 是一个特别有吸引力的 SARS-CoV-2 RNA 靶点。在这里，我们展示了 FSE 的 6.9 Å 分辨率冷冻电镜结构（88 个核苷酸，约 28 kDa），并通过 RNA 纳米结构标记方法进行了验证。三级结构呈现出拓扑复杂的折叠，其中 5' 端穿过三茎假结内形成的环。在这种结构的指导下，我们开发了反义寡核苷酸，可在移码测定中损害 FSE 功能，并以 100 nM 浓度抑制 A549-ACE2 细胞中 SARS-CoV-2 病毒的复制。

更新日期：2021-08-23

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