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

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.

Layer-by-Layer Encapsulation of Herbicide-Degrading Bacteria for Improved Surface Properties and Compatibility in Soils.

E. coli cells overexpressing the enzyme atrazine chlorohydrolase were coated using layer-by-layer self-assembly. The polymeric coating was designed to improve the surface properties of the cells and create positively charged, ecologically safe, bio-hybrid capsules that can efficiently degrade the herbicide atrazine in soils. The physio-chemical properties of the bacteria/polymer interface were studied as a function of the polymeric composition of the shell and its thickness. Characterization of cell viability, enzyme activity, morphology, and size of the bio-capsules was done using fluorescence spectroscopy, BET and zeta potential measurements and electron microscopy imaging. Out of several polyelectrolytes, the combination of polydiallyldimethylammonium chloride and polysodium 4-styrenesulfonate improved the surface properties and activity of the cells to the greatest extent. The resulting bio-hybrid capsules were stable, well-dispersed, with a net positive charge and a large surface area compared to the uncoated bacteria. These non-viable, bio-hybrid capsules also exhibited a kinetic advantage in comparison with uncoated cells. When added to soils, they exhibited continuous activity over a six-week period and atrazine concentrations declined by 84%. Thus, the concept of layer-by-layer coated bacteria is a promising avenue for the design of new and sustainable bioremediation and biocatalytic platforms.