DNA Encoded Glycan Libraries as a next-generation tool for the study of glycan-protein interactions

Abstract

Interactions between glycans and glycan-binding proteins (GBPs) mediate diverse cellular functions, and therefore are of diagnostic and therapeutic significance. Current leading strategies for studying glycan-GBP interactions require specialized knowledge and instrumentation. In this study, we report a strategy for studying glycan-GBP interactions that uses PCR, qPCR and nextgeneration sequencing (NGS) technologies that are more routinely accessible. Our headpiece conjugation-code ligation (HCCL) strategy couples glycans with unique DNA codes that specify glycan sugar moieties and glycosidic linkages when sequenced. We demonstrate the technology by synthesizing a DNA encoded glycan library of 50 biologically relevant glycans (DEGL-50) and probing interactions against 25 target proteins including lectins and antibodies. Data show glycan-GPB interactions in solution that are consistent with lower content, lower throughput ELISA assays. Data further demonstrate how monovalent and multivalent headpieces can be used to increase glycan-GPB interactions and enrich signals while using smaller sample sizes.The flexibility of our modular HCCL strategy has potential for producing large glycan libraries, facilitating high content-high throughput glycan binding studies, and increasing access to lower cost glyco-analyses.

Measuring carbohydrate recognition profile of lectins on live cells using liquid glycan array (LiGA)

“…We demonstrated that LiGA can be constructed using pre-synthesized glycans 10 and customized using enzymatic modifications directly on phage. 12 Inspired by DNA-encoded libraries (DEL), DNA-displayed glycan libraries have been developed; 13–15 however, LiGA offers several advantages over such display of glycans on ‘naked’ DNA (i) The internal location of the DNA barcode inside M13 virion in LiGA protects this DNA in demanding biological environment and thus make it possible to investigate the association of LiGA with GBPs on live cells both in vitro and in vivo ; 10,12 (ii) Examples of display of glycans on DNA are either mandatory monovalent display on 5’ end of DNA strand, 13,14 or fixed trivalent display on DNA hairpin 15 whereas number of glycans on phage can be controlled from 20 to 1500 copies per 700 nm-long virion. This density can also be encoded in DNA, 10 and it has a significant effect on interactions of glycans with target GBPs.…”

“…13,14 To partially mitigate this issue, Wang and co-workers displayed three copies of glycans on a library of DNA hairpins. 15 Nevertheless, the exposed DNA barcode is a liability, as it could be degraded by nucleases in a complex biological milieu and interfere with the binding of GBPs. Viral particles such as Qβ have unique advantages such as monodisperse morphology and size; they have been modified to display multiple copies of glycans to study glycan-GBP interactions in vitro and in vivo.…”

Chemoenzymatic Synthesis of Genetically-Encoded Multivalent Liquid N-glycan Arrays

“…Compared to monovalent DNA-coded glycan arrays [40][41][42][43] , the LiGA strategy provides the ability to control and encode glycan density. LiGA can also probe interactions with cells both in in vitro and in vivo.…”

Chemoenzymatic Synthesis of Genetically-Encoded Multivalent Liquid N-glycan Arrays

“…These genetically-encoded libraries of structurally-diverse multivalent glycoconjugates are powerful probes of GBP–glycan interactions 34 . Compared to monovalent DNA-coded glycan arrays 35–38 , the LiGA strategy provides the ability to control and encode glycan density. LiGA can also probe interactions with cells both in in vitro and in vivo .…”