Efficient Screening of Glycan-Specific Aptamers Using a Glycosylated Peptide as a Scaffold

Abstract

Abnormal glycan structures are valuable biomarkers for disease states; the development of glycan-specific binders is thereby significantly important. However, the structural homology and weak immunogenicity of glycans pose major hurdles in the evolution of antibodies, while the poor availability of complex glycans also has extremely hindered the selection of anti-glycan aptamers. Herein, we present a new approach to efficiently screen aptamers toward specific glycans with a complex structure, using a glycosylated peptide as a scaffold. In this method, using peptideimprinted magnetic nanoparticles (MNPs) as a versatile platform, a glycopeptide tryptically digested from a native glycoprotein was selectively entrapped for positive selection, while a nonglycosylated analogue with an identical peptide sequence was synthesized for negative selection. Alternating positive and negative selection steps were carried out to guide the directed evolution of glycanbinding aptamers. As proof of the principle, the biantennary digalactosylated disialylated N-glycan A2G2S2, against which there have been no antibodies and lectins so far, was employed as the target. With the glycoprotein transferrin as a source of target glycan, two satisfied anti-A2G2S2 aptamers were selected within seven rounds. Since A2G2S2 is upregulated in cancerous liver cells, carboxyfluorescein (FAM)-labeled aptamers were prepared as fluorescent imaging reagents, and successful differentiation of cancerous liver cells over normal liver cells was achieved, which demonstrated the application feasibility of the selected aptamers. This approach obviated a tedious glycan preparation process and allowed favorable expose of the intrinsic flexible conformation of natural glycans. Therefore, it holds great promise for developing glycan-specific aptamers for challenging applications such as cancer targeting.

New Promises of Advanced Molecular Recognition: Bioassays, Single Cell Analysis, Cancer Therapy, and Beyond

“…Schematic illustration of (A) the BA‐MNPs and CE‐based hybrid SELEX approach; [ 38 ] (B) glycan‐imprinted MNP based SELEX approach for the selection of glycoprotein‐binding aptamer; [ 39 ] (C) epitope‐Imprinted MNP‐Based SELEX for Efficient in vitro evolution of protein‐binding aptamers; [ 40 ] (D) SELEX selection of glycan‐binding aptamers on peptide‐imprinted MNPs. [ 41 ] …”

“…Based on our epitope imprinted polymers, we presented a new approach to efficiently screen aptamers toward specific glycans with a complex structure. [ 41 ] In this method, peptide imprinted MNPs was used as a versatile platform. As illustrated in Scheme 8D, a glycopeptide digested from a native glycoprotein was selectively entrapped for positive selection, while a nonglycosylated analogue with an identical peptide sequence was synthesized for negative selection.…”

High Mannose-Specific Aptamers for Broad-Spectrum Virus Inhibition and Cancer Targeting

“…35,36 Particularly, flexible use of MIPs as potent affinity platforms for positive and negative selection has enabled efficient selection of glycan-specific aptamers. 37 The long pandemic of COVID-19 since early 2019 has particularly motivated us to develop high mannose-binding aptamers for anti-virus fight.…”

Hydrophilic magnetic covalent triazine frameworks for differential N-glycopeptides enrichment in breast cancer plasma membranes

“…To solve the problems of low abundance of PMGs and the abundant interfering biomolecules, enrichment of glycosylated molecules is necessary before identification and analysis. There are several methods for enrichment of glycosylated molecules: hydrazide chemistry, 14 lectin affinity chromatography, 15 the boric acid affinity technique, [16][17][18][19] and hydrophilic interaction liquid chromatography (HILIC). 20 Among them, HILIC is one of the most important methods for the enrichment of glycopeptides and has received widespread attention due to its high enrichment performance and simple operation.…”