Abstract

Glycans are present in all domains of life and have broad physiological functions that are implicated not only in normal, healthy biological functions, but also in various diseases such as cancer, making them diagnostic and therapeutic targets. The study of glycans is currently limited in part by a lack of specific tools to target glycans of interest, with available glycan binding proteins often having low affinity and specificity. Thus, there is a need to develop new tools that can accelerate the development of novel glycan binding proteins. Here we discuss the application of directed evolution by mRNA display using new binding protein scaffold libraries, in combination with molecular dynamics simulations, as tools for developing novel glycan binding proteins.
We designed mRNA display compatible libraries of affibodies and designed ankyrin repeat protein (DARPin) to be screened for binding against sialyl Lewis X (SLeX), a tumour associated carbohydrate antigen that is overexpressed on cell surfaces of various cancers. We developed an improved enzymatic synthesis protocol for SLeX and describe a click chemistry immobilization method coupled with a fluorescent-based lectin assay to test glycan immobilization. The affibody library was successfully created for future selection, whereas the DARPin library assembly needs to be optimized further. Future mRNA display selection should also be complemented with the molecular dynamics probing method developed here. We demonstrate that short, computationally inexpensive probing simulations were able to identify the binding site of a nucleoside sugar dTDP-Qui3N in an ankyrin domain of an N-formyltransferase. Future approaches could simulate protein variants selected by display methods to identify glycan-protein interactions that may be used to improve the protein design. In combination, the tools developed here provide a framework to accelerate the discovery and production of future glycan binding proteins with applications in cancer diagnostics and therapeutics.