PROJECT SUMMARY Nucleosomes are the basic building blocks of chromatin and composed of ~147bp DNA wrapped around a core histone octamer. Chromatin function is controlled by the dynamic addition / removal of histone post-translational modifications (PTMs). Significantly, alterations in specific PTMs are associated with changes in gene expression, driving the emergence / progression of many cancer pathologies. Mounting evidence indicates that screening chromatin modifiers in the context of nucleosomal substrates is essential to recapitulate specificity toward appropriate PTM sites, and thus biologically-relevant activity. However, PTM regulators continue to be studied via modified histone fragments, which poorly mimic chromatin regulation in vivo. EpiCypher is pioneering the commercial development of nucleosomes carrying disease-relevant PTMs (`designer nucleosomes' or `dNucs') for innovative drug discovery / development. The current leading technology to generate modified histones for dNuc assembly is native chemical ligation (NCL), which permits the scarless incorporation of diverse PTMs (e.g., methylation, acetylation, and phosphorylation) on a single histone subunit. However, NCL is a multistep and labor-intensive process that requires 6-8 weeks of dedicated effort to synthesize a single dNuc in the milligram scale. Therefore, a new methodology is greatly needed to support the rapid generation of dNucs with diverse PTM layouts. To meet this need, EpiCypher is joining with Dr. Matthew Levy from The Albert Einstein College of Medicine to develop a novel protein engineering tool for accelerated dNuc manufacturing. The Staphylococcus aureus Sortase A (SrtA) transpeptidase can be modified by directed evolution to alter its recognition sequence (LPXTG; where X = any amino acid) to seamlessly ligate `unnatural' protein substrates. Dr. Levy's group recently developed a powerful directed evolution approach to rapidly screen large (~1012) mutant libraries, generating a robust SrtA (SrtA-variant4) with improved activity over wild-type enzyme. In Aim 1, Dr. Levy's team will further evolve this highly active SrtA variant toward histone H3 to rapidly incorporate PTMs into this substrate. In Aim 2, EpiCypher will demonstrate how this tool can be used for accelerated dNuc manufacturing. First, we will optimize a single-step ligation reaction to synthesize large quantities of ultrapure dNucs for high-throughput assay development. We will also optimize ligation of modified peptides directly to pre-assembled nucleosomes, providing a powerful diversity- manufacturing platform capable of multiplex synthesis. In Phase 2, we will utilize the SrtA-H3 variant developed here to scale up manufacturing of a diverse catalog of dNucs with disease associated PTMs. In addition, we will optimize multiplexed dNuc manufacturing to develop dNuc-based arrays for drug discovery. Finally, we will continue to generate new SrtA variants that selectively target histone H4. The breakout technology described herein will provide transformational opportunities for the development of next generation cancer therapeutics and epigenetic tool development.