The long term objectives of this proposal are to demonstrate the utility of a new class of high-specificity high-affinity proteins called Lectenz[unreadable] as research reagents for use in recognition of disease- or cancer-related glycosylation sites. The specific aims are to create a reagent that is pan-specific for the detection of the glycan f3-0-GlcNAc itself, as well as variations of this reagent that are able to detect f3-0-GIcNAc in the context of its disease- or cancer-specific peptide sequence (glycosylation site). The ability to rapidly confirm the presence of f3-0-GlcNAc in proteins and tissues without the need to turn to more-elaborate techniques would provide a powerful tool to delineate differentially glycosylated proteins and eventually establish correlations between f3-0-GIcNAc regulation and associated disease states, such as cancer. Using a combination of point mutagenesis, display library screening, and forefront computational methods, the human enzyme O-GIcNAcase (hOGA) will be converted into a high-specificity biosensor for its natural substrate f3-0-GIcNAc. Lectenz[unreadable] have several potential advantages over antibodies or aptamers, including predefined specificity for the target antigen, ease of preparation in a monovalent form, and (for human homologs) a low probability of in vivo toxicity. The innovative use of computational methods results in efficiency gains in the design and screening of the display library, lowering costs and increasing success rates.