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Cryo-electron Microscopy and Exploratory Antisense Targeting of the 28-kDa Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome
Link to bioRxiv paper:
http://biorxiv.org/cgi/content/short/2020.07.18.209270v1?rss=1
Authors: Zhang, K., Zheludev, I. N., Hagey, R. J., Wu, M. T.-P., Haslecker, R., Hou, Y. J., Kretsch, R., Pintilie, G. D., Rangan, R., Kladwang, W., Li, S., Pham, E. A., Bernardin, C., Baric, R. S., Sheahan, T. P., Dsouza, V., Glenn, J. S., Chiu, W., Das, R.
Abstract:
Drug discovery campaigns against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are beginning to target the viral RNA genome1, 2. The frameshift stimulation element (FSE) of the SARS-CoV-2 genome is required for balanced expression of essential viral proteins and is highly conserved, making it a potential candidate for antiviral targeting by small molecules and oligonucleotides3-6. To aid global efforts focusing on SARS-CoV-2 frameshifting, we report exploratory results from frameshifting and cellular replication experiments with locked nucleic acid (LNA) antisense oligonucleotides (ASOs), which support the FSE as a therapeutic target but highlight difficulties in achieving strong inactivation. To understand current limitations, we applied cryogenic electron microscopy (cryo-EM) and the Ribosolve7 pipeline to determine a three-dimensional structure of the SARS-CoV-2 FSE, validated through an RNA nanostructure tagging method. This is the smallest macromolecule (88 nt; 28 kDa) resolved by single-particle cryo-EM at subnanometer resolution to date. The tertiary structure model, defined to an estimated accuracy of 5.9 [A], presents a topologically complex fold in which the 5' end threads through a ring formed inside a three-stem pseudoknot. Our results suggest an updated model for SARS-CoV-2 frameshifting as well as binding sites that may be targeted by next generation ASOs and small molecules.
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By Multimodal LLCLink to bioRxiv paper:
http://biorxiv.org/cgi/content/short/2020.07.18.209270v1?rss=1
Authors: Zhang, K., Zheludev, I. N., Hagey, R. J., Wu, M. T.-P., Haslecker, R., Hou, Y. J., Kretsch, R., Pintilie, G. D., Rangan, R., Kladwang, W., Li, S., Pham, E. A., Bernardin, C., Baric, R. S., Sheahan, T. P., Dsouza, V., Glenn, J. S., Chiu, W., Das, R.
Abstract:
Drug discovery campaigns against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are beginning to target the viral RNA genome1, 2. The frameshift stimulation element (FSE) of the SARS-CoV-2 genome is required for balanced expression of essential viral proteins and is highly conserved, making it a potential candidate for antiviral targeting by small molecules and oligonucleotides3-6. To aid global efforts focusing on SARS-CoV-2 frameshifting, we report exploratory results from frameshifting and cellular replication experiments with locked nucleic acid (LNA) antisense oligonucleotides (ASOs), which support the FSE as a therapeutic target but highlight difficulties in achieving strong inactivation. To understand current limitations, we applied cryogenic electron microscopy (cryo-EM) and the Ribosolve7 pipeline to determine a three-dimensional structure of the SARS-CoV-2 FSE, validated through an RNA nanostructure tagging method. This is the smallest macromolecule (88 nt; 28 kDa) resolved by single-particle cryo-EM at subnanometer resolution to date. The tertiary structure model, defined to an estimated accuracy of 5.9 [A], presents a topologically complex fold in which the 5' end threads through a ring formed inside a three-stem pseudoknot. Our results suggest an updated model for SARS-CoV-2 frameshifting as well as binding sites that may be targeted by next generation ASOs and small molecules.
Copy rights belong to original authors. Visit the link for more info