Summary Thousands of intracellular proteins are dynamically modified by monosaccharides of O-linked N- acetylglucosamine (O-GlcNAc). The cycling of O-GlcNAc is regulated by the concerted actions of enzymes encoded by just two genes: the O-GlcNAc transferase (OGT) and the O-GlcNAcase (OGA) that add and remove O-GlcNAc, respectively (1-4). Dysregulation of O-GlcNAc cycling, or levels of the nucleotide sugar used by OGT (UDP-GlcNAc), exacerbates the pathophysiology of a host of diseases including type II diabetes, cancer, neurodegeneration, heart failure, hypertension and aging (5-7). While technological and methodological innovations have improved our ability to detect, modulate, and site-map O-GlcNAc (8-13), many of these techniques have not been adopted by the broader scientific community thus inhibiting a mechanistic understanding of the roles that O-GlcNAc plays in potentiating disease. Often, the aforementioned approaches require specialized equipment and reagents, lack the specificity required to study a modification that is cycled by just two enzymes, or lead to significant off-target effects (10, 14-20). Thus, the goal of the studies proposed herein is to generate facile, inexpensive, chemical genetic tools that probe the roles of O-GlcNAc in vitro and in vivo. Specifically, we will address the following aims: Specific Aim #1: Enabling tunable, reversible chemical-genetic regulation of O-GlcNAcylation in an organelle-specific manner. These tools will combine destabilization domain (21-23) and nanotrap (24) technology to enable researchers to modulate the expression of OGT and OGA, as well as key enzymes within the hexosamine biosynthetic pathway, in a spatial, temporal, and dose-dependent manner. These tools will be applied to understanding the role of O-GlcNAc in prostate cancer. Specific Aim 2: Enabling tissue- and organelle-specific tagging of O-GlcNAc-modified proteins. This tool will harness the power of proximity Biotin ligation (25, 26) to enable tagging and enrichment of O- GlcNAcylated proteins in a cell-specific or organelle-specific manner. Together, the tools described in this proposal will overcome current experimental limitations associated with studying O-GlcNAc and facilitate studies focused on determining the role of O-GlcNAc at a mechanistic level in a broad range of models.