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Single Quantum Dot-Based Nanosensor for Sensitive Detection of O‑GlcNAc Transferase Activity
journal contribution
posted on 2017-11-08, 00:00 authored by Juan Hu, Yueying Li, Ying Li, Bo Tang, Chun-yang ZhangProtein
glycosylation is a ubiquitous post-translational modification
that plays crucial roles in modulating biological recognition events
in development and physiology. Human O-GlcNAc transferase (OGT) is
an intracellular enzyme responsible for O-linked N-acetylglucosamine (O-GlcNAc) glycosylation, and the deregulation
of OGT activity occurs in cancer, diabetes, and neurodegenerative
disease. Here we develop a single quantum dot (QD)-based nanosensor
for sensitive OGT assay. We design a Cy5/biotin-modified peptide with
a serine hydroxyl group for sensing OGT and a protease site adjacent
to the glycosylation site for proteinase cleavage, with a universal
nonradioactive UDP-GlcNAc as the sugar donor and a Cy5/biotin-modified
peptide as the substrate. In the presence of OGT, it catalyzes the
glycosylation reaction to generate a glycosylated peptide that is
a protease-protection peptide. The resultant glycosylated Cy5/biotin-modified
peptides may assemble on the surface of the streptavidin-coated QD
to obtain a QD–peptide–Cy5 nanostructure in which the
fluorescence resonance energy transfer (FRET) from the QD to Cy5 can
occur, leading to the emission of Cy5 which can be quantified by single-molecule
detection. This method exhibits high sensitivity with a limit of detection
of 3.47 × 10–13 M, and it is very simple and
straightforward without the involvement of any enzyme purification,
radioisotope-labeled sugar donors, specific antibodies, and the synthesis
of fluorescent UDP-GlcNAc analogues. Moreover, this method can be
used for enzyme kinetic analysis, quantitative detection of cellular
OGT activity, and the screening of OGT inhibitors, holding great potential
for further application in drug discovery and clinical diagnosis.