生物大分子如蛋白质、核酸、糖脂等的动态化学修饰与病理变化的关系是当今生物医学研究最为重要的前沿领域之一。细胞内蛋白质的动态O-GlcNAc糖基化与肿瘤等慢性疾病的发生发展密切相关，而其中很多机理尚未有明确的研究。因此，本项目拟以结直肠癌细胞中β-连环蛋白的O-GlcNAc糖基化为研究对象，利用mRNA展示技术定向筛选针对其N端O-GlcNAc糖基化和其C端的纳米抗体，通过将纳米抗体和黄色荧光蛋白相融合构建基于双分子荧光互补策略的荧光探针，以实现对活细胞内β-连环蛋白动态O-GlcNAc糖基化的荧光研究，并进一步探索β-连环蛋白O-GlcNAc糖基化与结直肠癌发生发展的关系。本研究首次基于纳米抗体发展特定蛋白质O-GlcNAc糖基化的选择性荧光探针，能为实现基于异常O-GlcNAc糖基化的临床病理分子诊断、靶向干预治疗提供理论基础，对最终提高肿瘤的精准诊治与预防的效益极其重要。

The development and progression of many diseases are mostly associated with the dynamic chemical modifications of bio-macromolecules such as protein, nuclear acid, and glycolipid, so the relationship between a disease state and the abnormal chemical modification of bio-macromolecules has been one of the focused areas of the frontier biomedical research in these years. The posttranslational modification of proteins with O-GlcNAc is one of the most important dynamic chemical modifications in cells and the abnormal O-GlcNAcylation has been linked to chronic diseases such as cancer, but in many cases the behind mechanisms are not fully understood yet. Hence, the present project is aimed to develop a Nanobody-based fluorescent probe for visualizing the dynamic O-GlcNAcylation of β-catenin in colorectal cancer. First, we will identify specific Nanobodies against β-catenin C terminus and its O-GlcNAcylated N terminus by screening a large library using mRNA display. Second, we will construct the planed fluorescent probe by fusing the selected Nanobodies to the YFP-based bimolecular fluorescence complementation (BiFc) system. Finally, with this novel probe we will study the role of the dynamic O-GlcNAcylation of β-catenin in colorectal cancer development. To our knowledge, this is the first attempt to develop a novel probe for O-GlcNAc using Nanobodies and we believe that the final probe will further help study the cellular function of the dynamic O-GlcNAcylation in living cells. In addition, the related studies will also provide basis for developing the O-GlcNAcylation-based molecular diagnostic reagents and the target-specific therapeutic strategy.