Technologies for Directed Evolution of Glycoaptamers

项目摘要/摘要 这个聚焦技术R01项目的目标是开发用于快速发现的技术 碳水化合物结合蛋白(CBPS)的抑制剂。碳水化合物与蛋白质的相互作用是 在许多过程中至关重要，包括宿主-病原体识别、细胞黏附和细胞 信号，癌症血管生成，免疫抑制，心/肝/肺/眼纤维化， 动脉粥样硬化、糖尿病和类风湿性关节炎，仅举几例。然而，有选择性的 在体内，破坏特定的CBP相互作用是具有挑战性的。CBPS通常弱结合到 单个多糖，具有更高的亲和力(亲和力)或来自多价相互作用的特异性 到聚集的多糖或与附近的非碳水化合物元素的相互作用。一个给定的CBP也 通常与各种糖链结合。要理清CBP职能的复杂网络，重要的是 有一种方法可以快速开发出一种特定而有效的CBP抑制剂，这种抑制剂可以用于 与活体环境相关。为此，我们提出了两个目标：1)研制多价糖F-RNA 适配子，其中一个抗核酸酶的F-RNA骨架被进化成呈现简单的多糖 以被感兴趣的CBP选择性识别的方式；2)开发单价糖基- F-RNA适配子，其中一个简单的多糖为目的CBP提供结合的“立足点”， 进化的F-RNA成分通过以下方式赋予额外的结合亲和力和特异性 糖链结合口袋的辅助相互作用。

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.