Cryo-electron Microscopy and Exploratory Antisense Targeting of the 28-kDa Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome

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.

doi:10.1101/2020.07.18.209270

bioRxiv · 10.1101/2020.07.18.209270

Cryo-electron Microscopy and Exploratory Antisense Targeting of the 28-kDa Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome

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.

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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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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.. 2020-07-20. Cryo-electron Microscopy and Exploratory Antisense Targeting of the 28-kDa Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome. https://doi.org/10.1101/2020.07.18.209270

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