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A cell-free assay for rapid screening of inhibitors of hACE2-receptor - SARS-CoV-2-Spike binding

Nanami Kikuchi, Or Willinger, Naor Granik, Noa Navon, Shanny Ackerman, Ella Samuel, Tomer Antman, Noa Katz, Sarah Goldberg, Roee Amit
doi: https://doi.org/10.1101/2021.10.06.462907
Nanami Kikuchi
‡Department of Biotechnology and Food Engineering, Technion - Israel Institute of Technology, Haifa 32000, Israel
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Or Willinger
‡Department of Biotechnology and Food Engineering, Technion - Israel Institute of Technology, Haifa 32000, Israel
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Naor Granik
‡Department of Biotechnology and Food Engineering, Technion - Israel Institute of Technology, Haifa 32000, Israel
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Noa Navon
†Department of Biomedical Engineering, Technion - Israel Institute of Technology, Haifa 32000, Israel
‡Department of Biotechnology and Food Engineering, Technion - Israel Institute of Technology, Haifa 32000, Israel
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Shanny Ackerman
‡Department of Biotechnology and Food Engineering, Technion - Israel Institute of Technology, Haifa 32000, Israel
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Ella Samuel
‡Department of Biotechnology and Food Engineering, Technion - Israel Institute of Technology, Haifa 32000, Israel
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Tomer Antman
‡Department of Biotechnology and Food Engineering, Technion - Israel Institute of Technology, Haifa 32000, Israel
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Noa Katz
‡Department of Biotechnology and Food Engineering, Technion - Israel Institute of Technology, Haifa 32000, Israel
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Sarah Goldberg
‡Department of Biotechnology and Food Engineering, Technion - Israel Institute of Technology, Haifa 32000, Israel
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Roee Amit
‡Department of Biotechnology and Food Engineering, Technion - Israel Institute of Technology, Haifa 32000, Israel
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  • For correspondence: roeeamit@technion.ac.il
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ABSTRACT

We present a cell-free assay for rapid screening of candidate inhibitors of protein binding, focusing on inhibition of the interaction between the SARS-CoV-2 Spike receptor binding domain (RBD) and human angiotensin-converting enzyme 2 (hACE2). The assay has two components: fluorescent polystyrene particles covalently coated with RBD, termed virion-particles (v-particles), and fluorescently-labeled hACE2 (hACE2F) that binds the v-particles. When incubated with an inhibitor, v-particle - hACE2F binding is diminished, resulting in a reduction in the fluorescent signal of bound hACE2F relative to the non-inhibitor control, which can be measured via flow cytometry or fluorescence microscopy. We determine the amount of RBD needed for v-particle preparation, v-particle incubation time with hACE2F, hACE2F detection limit, and specificity of v-particle binding to hACE2F. We measure the dose response of the v-particles to a known inhibitor. Finally, we demonstrate that RNA-hACE2F granules trap v-particles effectively, providing a basis for potential RNA-hACE2F therapeutics.

Competing Interest Statement

N. Kikuchi, O. W., N. G., S. G., and R. A. are inventors on US Provisional Patent Application No. 63/187969 concerning some of the described technologies. N. Katz and R. A. are inventors on US Patent Application 2021/0095296 A1.

  • ABBREVIATIONS

    RBD
    SARS-CoV-2 Spike receptor-binding domain
    hACE2
    human angiotensin-converting enzyme 2
    tdPP7
    tandem-dimer form of bacteriophage PP7 coat protein
    hACE2F
    fluorescently-labeled hACE2 also containing tdPP7
    slncRNA-PP7bsx14
    synthetic long noncoding RNA harboring 14 binding sites of bacteriophage PP7 coat-protein
  • Copyright 
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    Posted October 06, 2021.
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    A cell-free assay for rapid screening of inhibitors of hACE2-receptor - SARS-CoV-2-Spike binding
    Nanami Kikuchi, Or Willinger, Naor Granik, Noa Navon, Shanny Ackerman, Ella Samuel, Tomer Antman, Noa Katz, Sarah Goldberg, Roee Amit
    bioRxiv 2021.10.06.462907; doi: https://doi.org/10.1101/2021.10.06.462907
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    A cell-free assay for rapid screening of inhibitors of hACE2-receptor - SARS-CoV-2-Spike binding
    Nanami Kikuchi, Or Willinger, Naor Granik, Noa Navon, Shanny Ackerman, Ella Samuel, Tomer Antman, Noa Katz, Sarah Goldberg, Roee Amit
    bioRxiv 2021.10.06.462907; doi: https://doi.org/10.1101/2021.10.06.462907

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