Formation of synthetic RNA protein granules using engineered phage-coat-protein -RNA complexes

Granik, Naor; Katz, Noa; Goldberg, Sarah; Amit, Roee

doi:10.1101/682518

Cited by 2 publications

(2 citation statements)

References 72 publications

(150 reference statements)

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“…Here, the v-particles provide a safe and microscopically visible alternative to actual virions. RNA-protein granules can be produced in vitro , , and have been shown to bind cellular components. , We recently showed that SRNP granules form specifically in vitro via self-assembly by mixing purified bacterial phage coat proteins with synthetic long noncoding RNA (slncRNA) molecules that encode multiple binding sites for the coat proteins. In this case, the protein component in the granule formulation was either tdPP7-mCherry, or hACE2F (see Supplementary Methods and Supplementary Table 1 for both proteins).…”

Section: Resultsmentioning

confidence: 99%

A Cell-Free Assay for Rapid Screening of Inhibitors of hACE2-Receptor–SARS-CoV-2-Spike Binding

et al. 2022

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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 noninhibitor 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 known inhibitors. Finally, utilizing an RNA-binding protein tdPP7 incorporated into hACE2F, we demonstrate that RNA-hACE2F granules trap v-particles effectively, providing a basis for potential RNA-hACE2F therapeutics.

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Section: Resultsmentioning

confidence: 99%

A Cell-Free Assay for Rapid Screening of Inhibitors of hACE2-Receptor–SARS-CoV-2-Spike Binding

et al. 2022

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show abstract

“…RNA-protein granules can be produced in vitro 29,30 , and have been shown to bind cellular components 30,31 . We recently showed 32 that SRNP granules form specifically in vitro via self-assembly by mixing purified bacterial phage coat proteins with synthetic long non-coding RNA (slncRNA) molecules that encode multiple binding sites for the coat proteins. In this case, the protein component in the granule formulation was either tdPP7-mCherry, or hACE2F (see Supplementary Methods and Supplementary Table 1 for both proteins).…”

Section: Resultsmentioning

confidence: 99%

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

Kikuchi

Willinger

Granik

et al. 2021

Preprint

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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.

show abstract

Formation of synthetic RNA protein granules using engineered phage-coat-protein -RNA complexes

Cited by 2 publications

References 72 publications

A Cell-Free Assay for Rapid Screening of Inhibitors of hACE2-Receptor–SARS-CoV-2-Spike Binding

A Cell-Free Assay for Rapid Screening of Inhibitors of hACE2-Receptor–SARS-CoV-2-Spike Binding

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

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