The inventors have engineered proteins for detection and manipulation of carbohydrates and carbohydrate-containing biomolecules with applications in basic glycoscience research, diagnostics, and therapeutics.
Glycans are ubiquitous and essential biomolecules with diverse biological functions, including maintenance of cell structure, signal transduction, and cellular interactions. Furthermore, aberrant glycosylation is implicated in a wide range of human diseases, such as cancer, neuronal disease, and diabetes. However, further progress in glycan-related research and therapeutic application is significantly limited by the lack of analytical tools capable of recognizing these structurally diverse biomolecules with high affinity and specificity. Glycans are poorly immunogenic, hindering development of highly selective anti-glycan antibodies. Lectins have been used for recognition and purification of glycosylated biomolecules, but lectins have a very limited epitope range and modest affinity for carbohydrates. Therefore, there exists a strong need to develop reagents capable of binding glycans with high specificity and affinity.
The inventors have engineered proteins capable of recognizing carbohydrate epitopes for use as antibody and lectin alternatives. Using directed evolution, the inventors engineered small DNA-binding proteins to tightly bind and specifically recognize targeted monosaccharides, disaccharides and more complex carbohydrates. These proteins are highly thermostable, small enough to recognize single-atom differences between sugars, and do not face the steric hindrance challenges of antibodies and lectins. Importantly, multiple glycan-binding proteins can be assembled together to form customized reagents for recognition and manipulation of complex oligosaccharide structures. These engineered glycan-binding proteins can be easily functionalized and can substitute for antibodies in a wide range of immunological or diagnostic techniques, including flow cytometry and histology for cells expressing a glycosylated biomolecule of interest. Furthermore, these proteins can be utilized in microarray-based detection methods or for affinity purification of specific glycosylated biomolecules. In summary, these engineered glycan-binding proteins enable recognition and manipulation of carbohydrates of user interest, regardless of size and composition, with greater specificity and affinity than currently existing tools.
- Engineered glycan-binding proteins capable of recognizing a wide range of carbohydrates, regardless of size or composition, with greater specificity and affinity than antibodies and lectins
- Capable of detecting single-atom differences between carbohydrates, and not sterically hindered due to their small sizes
- Multiple glycan-binding proteins can be linked to specifically recognize and manipulate very complex oligosaccharide structures
- Can be easily functionalized for a broad range of biological techniques, including immunostaining and purification of glycosylated biomolecules and cells.