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Liberation of Protein-Specific Glycosylation Information for Glycan Analysis by Exonuclease III-Aided Recycling Hybridization

Author:
Chen, Yunlong, Ding, Lin, Song, Wanyao, Yang, Min, Ju, Huangxian
Source:
Analytical chemistry 2016 v.88 no.5 pp. 2923-2928
ISSN:
1520-6882
Subject:
drug therapy, fluorescein, glycosylation, hybridization, intergenic DNA, nucleotide sequences, oligonucleotides, sialic acids
Abstract:
A strategy for information liberation of protein-specific glycosylation is designed via an exonuclease III-aided recycling “hybridization and cleavage” process with glycan and protein probes, which achieves homogeneous quantification of cell surface glycan. The protein probe contains matching and spacer DNA sequences and an aptamer specific to target protein. The glycan probe contains a complementary sequence modified with neighboring fluorescein and quencher, a spacer sequence, and a dibenzocyclooctyne-amine end to bind azide-tagged glycan. Upon sequential binding to their targets, the complementary sequences of two probes approach enough for their hybridization, which leads to the cleavage of hybridized glycan probe by exonuclease III and followed recycling “hybridization and cleavage” process of protein probe with other adjacent glycan probes to release the labeled fluorescein for obtaining the information on protein-specific glycosylation. This protocol has been used to in situ quantify EpCAM-specific sialic acid on MCF-7 cell surface and monitor its variation during drug treatment. This work demonstrates a powerful quantification tool for research of glycosylation.
Agid:
5304537