The overall objective of this project is to develop boronic acid-containing peptide aptamer-based fluorescent proteins (BapaFPs) for the detection of vicinal diol-containing molecules, including various carbohydrates and carbohydrate derivatives. In particular, this three-year project is to engineer BapaFPs for live-cell imaging of nucleotide sugars that are signaling molecules and donors for the enzymatic formation of polysaccharides, glycoproteins, glycolipids, and glycosylated secondary metabolite. This new technology features a genetically encoded boronic acid moiety to induce initial interactions with vicinal diol-containing molecules, (an) extended peptide aptamer(s) to enhance affinity and specificity, and a circularly permuted red fluorescent protein (cpRFP) domain for fluorescence readouts. This work holds great promise to fill the technical gap to enable fluorescence imaging and tracking of various carbohydrates and carbohydrate derivatives in live cells. We will pursue the following specific aims: 1. Engineer BapaFPs to selectively detect nucleotide sugars. 2. Characterize BapaFPs in live mammalian cells. This project will lead to new research tools for imaging nucleotide sugars in live mammalian cells and subcellular domains. Since nucleotide sugars connect metabolism to signaling (e.g. posttranslational modifications (PTMs), epigenetics, exocytotic signaling, and surface interactions) and biological outcomes, these new tools will catalyze a large array of biological studies related to glycan-mediated signaling and disease. In addition, these new biosensors will accelerate the discovery of selective inhibitors for glycosyltransferases and glycan modification enzymes. Because the BapaFP technology is straightforward, cost-effective, and scalable, and can be adopted by any laboratory with basic molecular and cell biology knowledge, we expect the broad application of BapaFPs for the tracking, identification, and analysis of carbohydrates and carbohydrate derivatives.