PROJECT SUMMARY Enzymes that add (writers) or remove (erasers) histone post-translational modifications (PTMs) are high value drug targets with the potential to treat diverse human pathologies (e.g. cancer, neurodegeneration or inflammation). Modified histone peptides are widely used as in vitro substrates due to their short development cycle and low cost. However, nucleosomes (Nucs) represent the physiological substrate for chromatin writer / eraser enzymes and are thus preferable for the development of target-specific therapeutics. Recent advances in the commercial manufacturing of PTM-containing recombinant Nucs (aka. designer Nucleosomes or dNucs) have realized these substrates for epigenetic HTS assay development. There is a great unmet need for a universal HTS platform compatible with diverse epigenetic substrates (e.g. histone peptides or Nucs), and capable of quantifying the activity of writer and eraser enzymes. Indeed, many current HTS assays (e.g. FRET and FP) are incompatible with Nuc-based substrates due to their large size vs. histone peptides. Moreover, eraser assays provide a formidable challenge for the leading antibody-based HTS platforms (e.g. AlphaLISA and TRF), as antibodies that recognize specific unmodified histone residues are few and far between. This has hampered drug development programs to epigenetic erasers, despite the overwhelming evidence implicating these enzymes in disease initiation / progression. In this proposal, we will develop EpiBeaconTM, a novel non-radioactive, no-wash epigenetic HTS platform. This innovative HTS platform will: 1) be compatible with both peptide- and Nuc-based substrates; 2) mitigate the costly infrastructure and reagents required by existing epigenetic HTS systems; and 3) provide access to difficult drug targets (e.g. erasers). The cornerstone of our technology is the development of specialized RNA aptamers, which will be used as `no-wash' detection reagents. These aptamer beacons will be engineered to distinguish the precise status of specific histone residues (e.g. H4K20 vs. H4K20me1) and fluoresce only upon target binding. The innovation of this project is a unique selection strategy allied with the novel incorporation of hydrophobic residues to generate aptamer beacons that will bind PTM- status targets with exquisite specificity while being agnostic to substrate type (i.e. peptides or Nucs). In Phase I, we will develop aptamer beacons to unmodified H4K20 (H4K20me0) and H4K20me1 (Aim 1) and validate these reagents by optimizing enzymatic assays for two high interest drug targets: SETD8 (writer) and LSD1n (eraser) (Aim 2). In Phase II, we will synthesize / optimize new histone-targeting aptamers as well as miniaturize (1536-well format) SETD8 and LSD1n assays for pilot screen validation.