Ordered two-dimensional arrays such as S-layers 1 , 2 and designed analogues 3 – 5 have intrigued bioengineers, 6 , 7 but with the exception of a single lattice formed with flexible linkers, 8 they are constituted from just one protein component. For modulating assembly dynamics and incorporating more complex functionality, materials composed of two components would have considerable advantages. 9 – 12 Here we describe a computational method to generate co-assembling binary layers by designing rigid interfaces between pairs of dihedral protein building-blocks, and use it to design a p6m lattice. The designed array components are soluble at mM concentrations, but when combined at nM concentrations, rapidly assemble into nearly crystalline micrometer-scale arrays nearly identical (based on TEM and SAXS) to the computational design model in vitro and in cells without the need for a two-dimensional support. Because the material is designed from the ground up, the components can be readily functionalized, and their symmetry reconfigured, enabling formation of ligand arrays with distinguishable surfaces which we demonstrate can drive extensive receptor clustering, downstream protein recruitment, and signaling. Using AFM on supported bilayers and quantitative microscopy on living cells, we show that arrays assembled on membranes have component stoichiometry and structure similar to arrays formed in vitro, and thus that our material can impose order onto fundamentally disordered substrates like cell membranes. In sharp contrast to previously characterized cell surface receptor binding assemblies such as antibodies and nanocages, which are rapidly endocytosed, we find that large arrays assembled at the cell surface suppress endocytosis in a tunable manner, with potential therapeutic relevance for extending receptor engagement and immune evasion. Our work paves the way towards a synthetic cell biology, where a new generation of multi-protein macroscale materials is designed to modulate cell responses and reshape synthetic and living systems.

Angiopoietin 1 and 2 (Ang1 and Ang2) modulate angiogenesis and vascular homeostasis through engagement of their very similar F-domain modules with the Tie2 receptor tyrosine kinase on endothelial cells. Despite this similarity in the underlying receptor binding interaction, the two angiopoietins have opposite effects: Ang1 induces phosphorylation of protein kinase B (AKT), strengthens cell-cell junctions and enhances endothelial cell survival while Ang2 antagonizes these effects1–4. To investigate the molecular basis for the opposing effects, we examined the protein kinase activation and morphological phenotypes produced by a series of computationally designed protein scaffolds presenting the Ang1 F-domain in a wide range of valencies and geometries. We find two broad phenotypic classes distinguished by the number of presented F-domains: scaffolds presenting 4 F-domains have Ang2 like activity, upregulating pFAK and pERK but not pAKT, and failing to induce cell migration and tube formation, while scaffolds presenting 6 or more F-domains have Ang1 like activity, upregulating pAKT and inducing migration and tube formation. The scaffolds with 8 or more F-domains display superagonist activity, producing stronger phenotypes at lower concentrations than Ang1. When examined in vivo, superagonist icosahedral self-assembling nanoparticles caused significant revascularization in hemorrhagic brains after a controlled cortical impact injury.

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microRNAs are ~22bp nucleotide non-coding RNAs that play important roles in the post-transcriptional regulation of gene expression. Many studies have established that microRNAs are important for cell fate choices, including the naïve to primed pluripotency state transitions, and their intermediate state, the developmentally suspended diapause state in early development. However, the full extent of microRNAs associated with these stage transitions in human and mouse remain under-explored. By meta-analysis of microRNA-seq, RNA-seq, and metabolomics datasets from human and mouse, we found a set of microRNAs, and importantly, their experimentally validated target genes that show consistent changes in naïve to primed transitions (microRNA up, target genes down, or vice versa). The targets of these microRNAs regulate developmental pathways (e.g., the Hedgehog-pathway), primary cilium, and remodeling of metabolic processes (oxidative phosphorylation, fatty acid metabolism, and amino acid transport) during the transition. Importantly, we identified 115 microRNAs that significantly change in the same direction in naïve to primed transitions in both human and mouse, many of which are novel candidate regulators of pluripotency. Furthermore, we identified 38 microRNAs and 274 target genes that may be involved in diapause, where embryonic development is temporarily suspended prior to implantation to uterus. The upregulated target genes suggest that microRNAs activate stress response in the diapause stage. In conclusion, we provide a comprehensive resource of microRNAs and their target genes involved in naïve to primed transition and in the paused intermediate, the embryonic diapause stage.