Single Quantum Dot-Based Nanosensor for Sensitive Detection of O

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Single Quantum Dot-Based Nanosensor for Sensitive Detection of O-GlcNAc Transferase Activity Juan Hu, Yueying Li, Ying Li, Bo Tang, and Chun-yang Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04065 • Publication Date (Web): 08 Nov 2017 Downloaded from http://pubs.acs.org on November 9, 2017

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Analytical Chemistry

Single Quantum Dot-Based Nanosensor for Sensitive Detection of O-GlcNAc Transferase Activity Juan Hu,﹟,† Yueying Li,﹟,† Ying Li, ‡,† Bo Tang,﹟,* and Chun-yang Zhang﹟,* ﹟

College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of

Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University, Jinan 250014, China ‡

School of Medicine, Health Science Center, Shenzhen University, Shenzhen 518060, China

* Corresponding author. Tel.: +86 0531-86186033; Fax: +86 0531-82615258. E-mail: [email protected]. Tel.: +86 0531-86180010; Fax: +86 0531-86180017; [email protected].

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ABSTRACT: Protein 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.

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INTRODUCTION Protein glycosylation is a ubiquitous post-translational modification, with the proteins being modified by a single O-linked N-acetylglucosamine (O-GlcNAc) moiety at serine or threonine residues, termed O-GlcNAcylation.1 The O-GlcNAc modification plays critical roles in diverse cellular processes including signal transduction,2 gene expression,3 protein degradation,4 metabolism,5 circadian rhythms,6 and neurodegeneration.7 The O-GlcNAc transferase (OGT) is an intracellular enzyme responsible for the O-GlcNAc modification.8 The OGT enzyme catalyzes the transfer of a single O-GlcNAc moiety from UDP-GlcNAc to a serine or threonine residue in proteins for the generation of GlcNAc-β-Ser/Thr linkage,1,9,10 while the deregulation of OGT activity is associated with various diseases such as cancer,11-16 diabetes,17,18 and Alzheimer disease.19 Thus, OGT may become a potential therapeutic target for disease treatment.20,21 The OGT activity is usually measured by monitoring the transfer of a radiolabeled sugar from the sugar donor (e.g., UDP-[3H]-GlcNAc) to the protein substrate (e.g., Nup62).22 But the radio-labeled method involves the costly labeling reagents and the tedious separation procedure.22 To overcome these limitations, a variety of nonradioactive methods have been developed for OGT assay, including enzyme linked immunosorbent assay (ELISA),23 azide-ELISA,24 Ni-NTA plate immunoassay,25 fluorescent-based ligand displacement assay,26 protease-protection fluorescence resonance energy transfer (FRET) method,27 and protease-protection electrochemical assay.28 However，these methods involve the reduced efficiency as a result of transferring the solution-phase enzymatic reaction onto a microplate,24 the synthesis of phosphine-FLAG probe,24 the Staudinger ligation with phosphine probes,24,25 requirement of specific antibodies (e.g., glycopeptide-specific monoclonal antibodies, anti-FLAG antibody, and anti-TAMRA antibody),23-25 the complex chemical synthesis of fluorescent UDP-GlcNAc analogue,26 the complicated design of a FRET dye pair,27 and relatively poor sensitivity.28 In addition, OGT is temperature-sensitive and the loss of enzyme 3



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activity may occur during the enzyme purification,25 making it a great challenge to quantitatively detect cellular OGT activity in real samples. Therefore, the development of a simple and rapid method for quantitative OGT assay without the requirement of enzyme purification is highly desirable. The QDs are novel semiconductor nanocrystals with significant advantages of good photo-stability, high quantum yield, long fluorescence lifetime, broad absorption spectra from the ultraviolet to infrared region, narrow and size-tunable emission spectra, simultaneous excitation with a single light source, good resistance to chemical and photodegradation, and a stable surface for chemical modification,29-31 and have been widely used as the fluorophores32-37 the fluorescence resonance energy transfer (FRET) donors38-40 and acceptors in place of organic dyes.41,42 In contrast to the available organic dye donors, the use of the QDs as the FRET donors offers several unique spectroscopic properties, such as improved FRET efficiency resulting from multiple acceptors around a central QD, tunable spectral overlap between the acceptor and the QD, minimization of direct acceptor excitation, and multiplex FRET configurations.43 Herein, we develop a single QD-based nanosensor to sensitively detect OGT activity. In comparison with the ensemble measurement, single-molecule detection has distinct advantages of ultrahigh sensitivity, low sample consumption, rapidity and simplicity,44,45 and has been successfully applied for sensitive detection of DNAs,46 microRNAs,47 proteins48 and even cancer cells49 at the single-molecule level. We design a Cy5/biotin-modified peptide with one serine hydroxyl group for sensing OGT and a unique protease site adjacent to the glycosylation site for proteinase cleavage, with a universal nonradioactive UDP-GlcNAc as the sugar donor and the peptide as the substrate. When OGT is present, it catalyzes the glycosylation reaction to generate a glycosylated peptide that is a protease-protection peptide. The glycosylated Cy5/biotin-modified peptides can assemble on the surface of the streptavidin-coated QD to obtain a QD-peptide-Cy5 nanostructure, resulting in efficient FRET from the QD to Cy5 and consequently the emission of Cy5 which can be quantified by total internal reflection 4


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fluorescence (TIRF)-based single-molecule detection. This single-QD-based nanosensor can be further used for enzyme kinetic analysis, quantitative detection of cellular OGT activity and the screening of OGT inhibitors.

MATERIALS AND METHODS Materials. The recombinant human O-GlcNAc transferase (OGT) was purchased from R&D Systems (Minneapolis, MN, U.S.A.). The peptide was obtained from Chinese Peptide Company (Hangzhou, China), and

its

sequence

is

5′-diphospho-N-acetylglucosamine

Biotin-Ser-Thr-Pro-Val-Ser-Arg-Ala-Asn-Met-Lys-Cy5. sodium

salt

(UDP-GlcNAc),

proteinase

K

and

Uridine OSMI-1

((αR)-α-[[(1,2-Dihydro-2-oxo-6-quinolinyl)sulfonyl]amino]-N-(2-furanylmethyl)-2-methoxy-N-(2-thienylmet hyl)-benzeneacetamide) were purchased from Sigma-Aldrich Company (St. Luois, MO, U.S.A.). Alkaline phosphatase, CutSmart buffer (100 mM magnesium acetate, 200 mM Tris-acetate, 500 mM potassium acetate, 1 mg/mL bovine serum albumin (BSA), pH 7.9) were obtained from New England Biolabs (Ipswich, MA, U.S.A.). The 605 nm-emitting streptavidin-coated CdSe/ZnS QDs (Qdot 605 ITK) were purchased from Invitrogen Corporation (Carlsbad, CA, U.S.A.). In vitro OGT Assay. The in vitro OGT assay includes three steps. First, 20 µL of solution containing 2.4 µM Cy5/biotin-modified peptide, 96 µM UDP-GlcNAc, 0.05 U of alkaline phosphatase, different-concentration OGT and 10× reaction buffer (500 mM Tris, 200 mM CaCl2, 0.5% Tween-20, 0.5% NP-40, pH 7.8) were incubated at 37°C for 1 h. Second, proteinase K was added into the glycosylation reaction system with 50 ng/mL proteinase K in a total volume of 30 µL, followed by reaction at 55°C for 2 h. Third, 10 nM QDs was added to 30 µL of the reaction products in 100 µL of incubation buffer (10 mM (NH4)2SO4, 100 mM Tris-HCl, 3 mM MgCl2, pH 8.0), and incubated for 20 min at room temperature to obtain the QD-peptide-Cy5 5



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nanostructures. The fluorescence measurement was carried out by using a Hitachi F-7000 fluorescence spectrophotometer (Tokyo, Japan). Single-Molecule Detection. After 500-fold dilution of the reaction products with the imaging buffer (2.5 mM MgCl2, 1 mg/mL Trolox, 50 µg/mL BSA, 0.04% mg/mL catalase, 67 mM glycine-KOH, 0.4% (w/v) D-glucose, 1 mg/mL glucose oxidase, pH 9.4), 10 µL of sample was dropped onto the coverslip for TIRF imaging, with 488-nm laser (20 mW) being used for the excitation of QDs. A 100× oil immersion objective and an EMCCD (Photometrics, Evolve 512) with an exposure time of 500 ms were used to collect the photons emitted from Cy5 and the QD. The image J software was used to count the Cy5 from an imaging region of 500 × 500 pixels. Inhibition Assay. For OGT inhibition assay, different-concentration OSMI-1 was mixed with 250 nM OGT in the reaction solution and incubated for 60 min at 37°C. The relative activity of OGT (RA) was calculated based on eq. 1

(%) = × 100%

(1)

where N0 is the Cy5 counts without OGT, Nt is the Cy5 counts in response to 250 nM OGT, and Ni is the Cy5 counts in response to 250 nM OGT and OSMI-1. The IC50 value was measured based on the curve of the relative activity of OGT versus the concentration of OSMI-1. Cell Culture and Preparation of Cell Extracts. HEK-293 cells were cultured in 1% penicillin-streptomycin and 10% fetal bovine serum (FBS) at 37 °C in a humidified chamber containing 5% CO2. The cells were harvested by aspirating growth media and washing with ice cold 1× PBS, and then were lysed in 1% SDS / 20 mM HEPES (pH 7.9). Subsequently, 1 mM PMSF, 10 µM PUGNAC, 10 µM TMG and 1× complete protease inhibitor cocktail were added to the solution, followed by sonication. At last, the samples were centrifuged at 12000 rpm for 15 min, and the supernatant was collected for OGT activity assay immediately. 6


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RESULTS AND DISCUSSION Principle of OGT activity assay. The principle of OGT activity assay is illustrated in Scheme 1. We design a Cy5/biotin-modified peptide with one serine hydroxyl group for sensing OGT and a unique 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 this design, the proteinase cleavage site is the peptide bond adjacent to the carboxylic group of valine (i.e., the peptide bond between valine and serine), and the N-terminal biotin tag allows the peptide substrate to rapidly self-assemble on the surface of QD via biotin-streptavidin interaction. This assay contains three steps including (1) the O-linked glycosylation of Cy5/biotin-modified peptide by sugar donor UDP-GlcNAc, (2) the discrimination between glycosylated and non-glycosylated peptide by proteinase K, and (3) the construction of the QD-peptide-Cy5 nanostructure and the subsequent measurement of FRET signal (Scheme 1). In the presence of OGT enzyme, it catalyzes the transfer of single sugar GlcNAc from UDP-GlcNAc to the serine hydroxyl group of the peptide, resulting in the generation of a glycosylated peptide (see Supporting Information, Figure S1).9,10 The glycosylated peptide is a protease-protection peptide that can protect the polypeptide chain from the cleavage of proteinase K. Subsequently, the glycosylated Cy5/biotin-modified peptides may self-assembly onto the QD surface to obtain a QD-peptide-Cy5 nanostructure through specific biotin-streptavidin interaction, leading to efficient FRET from the QD to Cy5 and consequently the emission of Cy5 which can be simply measured for the quantification of OGT activity. While in the absence of OGT, the Cy5/biotin-modified peptide is unglycosylated and may be cleaved by proteinase K at the peptide bond between valine and serine,50 leading to the separation of Cy5 from the biotin. As a result, no Cy5 can be assembled onto the QD surface and no FRET occurs between the QD and Cy5. 7



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In this FRET-based assay, the 605 nm-emitting QD (605QD) functions as the donor with Cy5 as the acceptor on the basis of following factors: (1) the 605QD/Cy5 pair has significant 605QD emission/Cy5 absorption spectral overlap but negligible emission cross-talk,43 facilitating efficient FRET between them (see Supporting Information, Figure S2); (2) the 605QD/Cy5 pair has larger Förster distance (R0 = 77 Å, see Supporting Information),31 guaranteeing efficient FRET between them (When the polypeptide is maximally extended, each amino acid contributes 3.8 Å to the contour length,51 and the total peptide length of Cy5/biotin-modified peptide with 10 amino acids is estimated to be 38 Å. Taking into account the radius of a streptavidin-coated QD (50~75 Å), biotin (~3 Å)52 and Cy5 (1.5~11 Å),53 the largest donor-acceptor separation distance is calculated to be 130 Å, within the efficient FRET range (2R0 = 154 Å)); (3) multiple Cy5/biotin-modified

peptides

may

be

assembled

onto

a

single

605QD

to

obtain

a

single-donor/multiple-acceptor nanostructure for improved FRET efficiency;43 (4) Cy5 cannot be excited by the 488-nm laser, and the measured Cy5 signal results exclusively from FRET sensitization, ensuring the high sensitivity.

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Scheme 1. Schematic illustration of single QD-based nanosensor for OGT activity assay. This assay contains three steps including (1) the O-linked glycosylation of Cy5/biotin-modified peptide by sugar donor UDP-GlcNAc, (2) the discrimination between glycosylated and non-glycosylated peptide by proteinase K, and (3) the construction of the QD-peptide-Cy5 nanostructure and the subsequent measurement of FRET signal.

Validation of the Assay. We employed the fluorescence measurement to study the FRET from the QD to Cy5 (Figure 1). When OGT is absent, no Cy5/biotin-modified peptide can be assembled onto the QD surface, and no QD-peptide-Cy5 nanostructure can be obtained. Consequently, no significant Cy5 signal is observed (Figure 1, blue line). In contrast, the addition of OGT may initiate the O-linked glycosylation of Cy5/biotin-modified peptide to generate a protease-protection peptide which can assemble onto the surface of QD to obtain a QD-peptide-Cy5 nanostructure, resulting in efficient FRET from the QD to Cy5 and

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consequently the decrease of QD fluorescence and the increase of Cy5 fluorescence (Figure 1, red line). When 250 nM OGT is present, the FRET efficiency (E) is measured to be 76.5% based on eq. 2 =1

(2)

where FD is the QD fluorescence intensity in the absence of OGT, and FDA is the QD fluorescence intensity in the presence of OGT. For a single QD assembled by multiple acceptors, the FRET efficiency (E) is measured based on eq. 3 =

(3)

where E is FRET efficiency, n is the average number of acceptors, R0 is the Förster distance, and r is the average donor-acceptor separation distance. When E is 76.5%, R0 is 77 Å and n is 48, the average donor-acceptor separation distance of r was calculated to be 121 Å, which is reasonably consistent with the theoretical largest donor-acceptor separation distance of 130 Å in the QD-peptide-Cy5 nanostructure with the flexible peptide.

Figure 1. Measurement of Cy5 and QD fluorescence signals in response to the control without OGT (blue line) and 250 nM OGT (red line). The QD concentration is 10 nM.

In this research, TIRF microscopy was used for the imaging of the QD-peptide-Cy5 nanosensors. TIRF 10


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microscopy allows selective visualization of fluorophores within ~100 nm from the glass coverslip.54 On the basis of that the theoretical largest donor-acceptor separation distance is 130 Å, the largest diameter of the QD-peptide-Cy5 nanostructure is calculated to be 26 nm, within the excitation field of TIRF microscopy. When OGT is absent, only the QD fluorescence signals can be detected (Figure 2A, green color), without Cy5 fluorescence signal being observed (Figure 2B). When OGT is present, both the QD (Figure 2D, green color) and Cy5 (Figure 2E, red color) fluorescence signals can be observed simultaneously with perfect colocalization of both signals (Figure 2F, yellow color). Moreover, the QD fluorescence intensity in the presence of OGT (Figure 2D) is much lower than that without OGT (Figure 2A) due to efficient FRET from the QD to Cy5, consistent with the ensemble measurement result (Figure 1). These results demonstrate that OGT may induce efficient FRET from the QD to Cy5 and the quantification of Cy5 counts may be used for OGT assay.

Figure 2. Single-molecule fluorescence image in the absence (A-C) and presence of OGT (D-F). The QD fluorescence signal is shown in green (A, D), and the Cy5 fluorescence signal is shown in red (B, E). The yellow color indicates the colocalization of the QD and Cy5 (C, F). The scale bar is 8 µm.

Optimization of Experimental Conditions. To achieve the best performance, we optimized the peptide-to-QD ratio, the concentration of proteinase K, the UDP-GlcNAc-to-peptide ratio, and the incubation time, respectively. We first investigated the influence of the biotin/Cy5-modified peptide-to-QD ratio on the 11



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FRET efficiency. The biotin/streptavidin-linked QD nanostructure was formed through self-assembly of biotinylated peptides to the as-purchased streptavidin QDs.39,55 The 1 pmol of 605 nm streptavidin-coated QDs was mixed with 0, 6, 12, 24, 36, 42, 48, and 60 pmol of biotinylated peptides and the incubation buffer with a final QD concentration of 10 nM in total volume of 100 µL, followed by incubation at room temperature for 20 min prior to the measurement. After 500-fold dilution with the imaging buffer, the reaction products were subjected to TIRF imaging. In the single QD-based nanosensor, the FRET efficiency (E) can be calculated according to eq. 4 =1

=1

∑ ∑

(4)

where ΣIDA is the sum of QD fluorescence intensities in the presence of Cy5 acceptors, and ΣID is the sum of QD fluorescence intensities in the absence of Cy5 acceptors. As shown in Figure 3A, the FRET efficiency enhances with increasing peptide-to-QD ratio from 6 to 48, and reaches a plateau beyond the ratio of 48 (Figure 3A, blue line). Additionally, the Cy5 counts have a linear relationship with the peptide-to-QD ratio in the range from 6 to 48 (Figure 3A, red line), consistent with the optical bulk measurement result (see Supporting Information, Figure S3). The obtained peptide-to-QD ratio of 48:1 is consistent with the result of previous research (48:1),55 and is very close to the theoretically calculated biotin-binding sites per QD (36~45). Because of the assembly of 12~15 streptavidins per QD and 3 available biotin-binding sites per streptavidin,55 in theory there are up to 45 biotin-binding sites per QD. The minor discrepancy between the obtained peptide-to-QD ratio and the theoretically calculated biotin-binding sites per QD might be attributed to the difference in the synthesis of streptavidin-conjugated QD from batch to batch. Thus, the addition of peptide-to-QD ratio of 48 is used in the subsequent research. The key to this assay is the successful O-linked glycosylation of Cy5/biotin-modified peptide and the subsequent proteinase cleavage that discriminates between glycosylated and non-glycosylated peptides. We 12


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further investigated the influence of proteinase K concentration on the assay performance using the unglycosylated Cy5/biotin-modified peptide. The Cy5 counts decrease with the increasing concentration of proteinase K from 0.1 to 50 ng/mL, and reaches a plateau beyond the concentration of 50 ng/mL (Figure 3B) due to the complete cleavage of the unglycosylated Cy5/biotin-modified peptide by proteinase K. Thus, 50 ng/mL proteinase K is used in the subsequent research. We utilized a commercially available UDP-GlcNAc as the sugar donor (see Supporting Information, Figure S1) to optimize the UDP-GlcNAc-to-peptide ratio with the concentration of biotin/Cy5-modified peptide being fixed at 2.4 µM. The Cy5 counts enhance with the increasing UDP-GlcNAc-to-peptide ratio from 5 to 40, and reaches a plateau beyond the ratio of 40 (Figure 3C) due to either the complete loss of OGT activity or the consumption of all available substrate peptides by OGT. Thus, the UDP-GlcNAc-to-peptide ratio of 40 is used in the subsequent research. We further optimize the incubation time of O-linked glycosylation reaction. The Cy5 counts improve with the reaction time from 5 to 60 min, and reaches a plateau beyond 60 min (Figure 3D) due to either the complete loss of OGT activity or the consumption of all available substrates by OGT. Thus, the incubation time of 60 min is used for O-linked glycosylation reaction in the subsequent research.

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Figure 3. (A) Variance of FRET efficiency and Cy5 counts with the peptide-to-QD ratio measured by single-molecule detection. (B) Measurement of Cy5 counts in response to different proteinase K concentration. (C) Measurement of Cy5 counts in response to different UDP-GlcNAc-to-peptide ratio. (D) Influence of the reaction time of O-linked glycosylation reaction upon the measured Cy5 counts. Error bars represent the standard deviation of 3 independent experiments.

Detection Sensitivity. Under the optimal reaction conditions, we evaluated the detection sensitivity (Figure 4). When the OGT concentration increases from 4.0 × 10-13 to 2.5 × 10-7 M, the Cy5 counts increase correspondingly. In the logarithm scale, the Cy5 counts show a linear correlation with the OGT concentration 14


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from 4.0 × 10-13 to 2.5 × 10-10 M (inset of Figure 4). The regression equation is N = 2251.1 + 172.8 log10 C (R2 = 0.986), where C is the OGT concentration and N is the Cy5 counts, respectively. The limit of detection is calculated to be 3.47 × 10-13 M by evaluating the control group plus 3 times standard deviation. The sensitivity of this method has enhanced by as much as 4 orders of magnitude as compared with that of electrochemical assay (10 nM).28 The enhanced sensitivity can be ascribed to (1) the specific OGT-induced glycosylation of Cy5/biotin-modified peptide and subsequent proteinase K cleavage that can discriminate between glycosylated and non-glycosylated peptides,9,10 (2) the improved FRET efficiency resulting from the assembly of multiple Cy5/biotin-modified peptides on the surface of a single QD,43 and (3) the high sensitivity of single-molecule detection.44

Figure 4. Variance of the measured Cy5 counts with the OGT concentration. The inset shows the logarithm dependence of Cy5 counts upon the OGT concentration. Error bars represent the standard deviation of 3 independent experiments.

Detection Selectivity. To investigate the selectivity of the proposed method, we used bovine serum albumin (BSA) and immunoglobulin G (IgG) as the nonspecific proteins and three transferases including DNA adeninemethyltransferase (Dam; it catalyzes the transfer of a methyl group from the S-adenosylmethionine to 15



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adenine/cytosine residues), T4 polynucleotide kinase (PNK; it catalyzes the transfer of the terminal phosphate group from adenosine triphosphate (ATP) to the 5’-hydroxyl group of oligonucleotides), and cAMP-dependent protein kinase (PKA; it catalyzes the transfer of terminal phosphate from ATP to threonine/serine residues). In the presence of nonspecific proteins and other transferases that cannot recognize the biotin/Cy5-modified peptide substrate, the peptide substrate will be completely cleaved by proteinase K due to the lack of protection of GlcNAc glycosylated serine, resulting in the separation of Cy5 from the biotin. As a result, no Cy5 can be assembled on the QD surface and no FRET occurs. When OGT is present, it catalyzes the glycosylation reaction to produce a protease-protection peptide which can assemble on the surface of QD to obtain a QD-peptide-Cy5 nanostructure, resulting in the FRET from the QD to Cy5. Consequently, the Cy5 counts measured in the presence of OGT are much more than those measured in the presence of nonspecific proteins (i.e., BSA and IgG) and other transferases (i.e, Dam, PNK and PKA) (Figure 5), suggesting the high selectivity of the proposed method towards OGT.

Figure 5. The measured Cy5 counts in response to 250 nM OGT (red column), 250 nM BSA (green column), 250 nM IgG (blue column), 40 U/mL Dam (cyan column), 40 U/mL PNK (magenta column), 100 U/mL PKA (yellow column), and the reaction buffer (control). Error bars represent the standard deviation of 3 independent experiments.

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Kinetic Analysis. For kinetic analysis, we measured the initial velocity in the presence of 250 nM OGT and variable-concentration UDP-GlcNAc at 37 °C in 10 min reaction. When ~ 80% of the substrate (i.e., UDP-GlcNAc) is unconsumed, the reaction is in the initial rate regime.26,56 The enzymatic initial velocity is calculated as the number of consumed UDP-GlcNAc per minute based on the concentration of consumed UDP-GlcNAc (see Supporting Information, Figure S4) and the reaction time. Figure 6 shows that the initial velocity of OGT enhances with the increasing concentration of UDP-GlcNAc. We fit the experimental data to the Michaelis−Menten equation V = Vmax [S] / (Km + [S]), where Vmax is the maximum initial velocity, and [S] is the UDP-GlcNAc concentration, and Km is the Michaelis-Menten constant corresponding to the concentration at half-maximal velocity. The Vmax of OGT is evaluated to be 987.35 nM/min, and Km is calculated to be 19.01 µM. The Kcat value is calculated to be 3.95 min-1 based on the equation Kcat = Vmax/[enzyme], where [enzyme] is the concentration of OGT. The Km value is consistent with that obtained by the azido-ELISA method (22 µM),24 suggesting that this method can be used to accurately evaluate the kinetic parameters of OGT.

Figure 6. Measurement of initial velocity (V) versus the concentration of UDP-GlcNAc for kinetic analysis. Error bars represent the standard deviation of 3 independent experiments. 17



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Inhibition Assay. For inhibition assay, we used OSMI-1 as the OGT inhibitor.57 OSMI-1 is a cell-permeable OGT inhibitor with higher specificity against OGT than other extant compounds,57 but it minimally affects cell surface N- or O-linked glycans. Figure 7 shows the decrease of the relative activity of OGT as a function of the OSMI-1 concentration. We used the IC50 value (half-maximal inhibitory concentration) to evaluate the inhibition effect of OSMI-1 upon OGT enzyme. The IC50 value of OGT is calculated to be 3.05 µM, consistent with that measured by immunoblotting assay (2.7 µM).57 These results indicates that this method can be used to screen the OGT inhibitors.

Figure 7. Decrease of the relative activity of OGT as a function of the OSMI-1 concentration. Error bars represent the standard deviation of 3 independent experiments.

Detection of OGT from Cell Extracts. To demonstrate the feasibility of the proposed method for cellular OGT assay, we used human embryonic kidney cell line (HEK-293 cells) as the model.57 Figure 8 shows the increase of Cy5 counts as a function of the number of HEK-293 cells. In the logarithm scale, the Cy5 counts exhibit a linear correlation with the number of HEK-293 cells in the range from 5 to 10000 cells, and the corresponding equation is N = 346.12 log10 X - 112.52 (R2 = 0.992), where X is the number of HEK-293 cells 18


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and N is the Cy5 counts, respectively. The limit of detection is estimated to be 4 cells by calculating the control group plus three times standard deviation (see Supporting Information, Figure S5). To the best of our knowledge, this is the first report about the quantitative measurement of cellular OGT activity in real samples.

Figure 8. Logarithm dependence of Cy5 counts upon the number of HEK-293 cells from 5 to 10000 cells. Error bars represent the standard deviation of 3 independent experiments.

CONCLUSION In conclusion, we demonstrate the development of a single QD-based FRET sensor 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. Taking advantage of (1) the specific OGT-induced glycosylation of Cy5/biotin-modified peptide and the subsequent proteinase K cleavage that can distinguish between glycosylated and non-glycosylated peptides,9,10 (2) the improved FRET efficiency resulting from the assembly of multiple Cy5/biotin-modified peptides on the surface of a single QD,,43 and (3) the high sensitivity of single-molecule detection,44 the proposed method exhibits a large dynamic range from 4.0 × 10-13 to 2.5 × 10-10 M and extremely high sensitivity with a limit of detection of 3.47 × 10-13 M. To the 19



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best of our knowledge, this is the first report about the quantitative measurement of cellular OGT activity. Moreover, this method can accurately evaluate the enzyme kinetic parameters and screen the OGT inhibitors, with great potential applications in drug discovery and clinical diagnosis. Furthermore, this method may function as a universal sensing platform to detect other glycosyltransferases through the design of appropriate peptide substrates.58

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge available free of charge on the ACS Publications website at DOI: Mechanism of the protein glycosylation (Figures S1), analysis of FRET data (Figures S2), influence of substrate-to-QD ratio on FRET efficiency in the ensemble measurement (Figures S3), conversion equation (Figures S4), detection of OGT from cell extracts (Figures S5). (PDF). AUTHOR INFORMATION Corresponding Author *Tel.: +86 0531-86186033. Fax: +86 0531-82615258. E-mail: [email protected]. . *Tel.: +86 0531-86180010. Fax: +86 0531-86180017. E-mail: [email protected]. Author Contributions † These authors contributed equally. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT 20


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This work was supported by the National Natural Science Foundation of China (Grant Nos. 21325523, 31400655, 21527811, 21735003 and 21575152), and the Award for Team Leader Program of Taishan Scholars of Shandong Province, China.

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For TOC only

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Single Quantum Dot-Based Nanosensor for Sensitive Detection of O

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