Discovery of indole-modified aptamers for highly specific recognition of protein glycoforms

Glycosylation is one of the most abundant forms of post-translational modification, and can have a profound impact on a wide range of biological processes and diseases. Unfortunately, efforts to characterize the biological function of such modifications have been greatly hampered by the lack of affinity reagents that can differentiate protein glycoforms with robust affinity and specificity. In this work, we use a fluorescence-activated cell sorting (FACS)-based approach to generate and screen aptamers with indole-modified bases, which are capable of recognizing and differentiating between specific protein glycoforms. Using this approach, we were able to select base-modified aptamers that exhibit strong selectivity for specific glycoforms of two different proteins. These aptamers can discriminate between molecules that differ only in their glycan modifications, and can also be used to label glycoproteins on the surface of cultured cells. We believe our strategy should offer a generally-applicable approach for developing useful reagents for glycobiology research.

Discovery of indole-modified aptamers for highly specific recognition of protein glycoforms

Glycosylation is one of the most abundant forms of post-translational modification, and can have a profound impact on a wide range of biological processes and diseases. Unfortunately, efforts to characterize such modifications in the context of basic and clinical research are severely hampered by the lack of affinity reagents that can differentiate protein glycoforms. This lack of reagents is largely due to the challenges associated with generating affinity reagents that can bind to particular glycan epitopes with robust affinity and specificity. In this work, we use a fluorescence-activated cell sorting (FACS)-based approach to generate and screen aptamers with indole-modified bases in an effort to isolate reagents that can differentiate between protein glycoforms. Using this approach, we were able to select multiple aptamers that exhibit strong selectivity for specific glycoforms of two different proteins, with the capacity to discriminate between molecules with identical tertiary structures that differ only in terms of their glycan modifications.

Elucidating the role of carbohydrate determinants in regulating hemostasis: insights and opportunities

Recent improvement in modern analytical technologies has stimulated an explosive growth in the study of glycobiology. In turn, this has lead to a richer understanding of the crucial role of N- and O-linked carbohydrates in dictating the properties of the proteins to which they are attached and, in particular, their centrality in the control of protein synthesis, longevity, and activity. Given their importance, it is unsurprising that both gross and subtle defects in glycosylation often contribute to human disease pathology. In this review, we discuss the accumulating evidence for the significance of glycosylation in mediating the functions of the plasma glycoproteins involved in hemostasis and thrombosis. In particular, the role of naturally occurring coagulation protein glycoforms and inherited defects in carbohydrate attachment in modulating coagulation is considered. Finally, we describe the therapeutic opportunities presented by new insights into the role of attached carbohydrates in shaping coagulation protein function and the promise of carbohydrate modification in the delivery of novel therapeutic biologics with enhanced functional properties for the treatment of hemostatic disorders.