Technologies for Directed Evolution of Glycoaptamers

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Project Summary/Abstract The goal of this Focused Technology R01 project is to develop technologies for rapid discovery of inhibitors of carbohydrate-binding proteins (CBPs). Carbohydrate-protein interactions are critical in numerous processes including host-pathogen recognition, cell adhesion and cell signaling, cancer angiogenesis, immune suppression, heart/liver/lung/eye fibrosis, atherosclerosis, diabetes, and rheumatoid arthritis, to name a few examples. However, selective disruption of specific CBP interactions is challenging in vivo. CBPs generally bind weakly to individual glycans, with higher affinity (avidity) or specificity coming from multivalent interactions to clustered glycans or interactions with nearby non-carbohydrate elements. A given CBP also usually binds to various glycans. To untangle the complex web of CBP functions, it is important to have a way to rapidly develop a specific and potent inhibitor of a CBP that can be used in the relevant in vivo context. To this end, we propose two Aims: 1) to develop multivalent glyco-F-RNA aptamers, in which a nuclease-resistant, F-RNA backbone is evolved to present simple glycans in a manner that is selectively recognized by a CBP of interest; 2) to develop monovalent glyco- F-RNA aptamers, in which a simple glycan provides a binding “foothold” for the CBP of interest, which the evolved F-RNA component confers additional binding affinity and specificity through interactions auxiliary to the glycan-binding pocket.