Negative Regulation of Wnt/β-catenin Signaling is blocked by Wnt Signaling Component Protein Dishevelled1 Binding Aptamer (WD-Aptamer)

Construct Design, Protein Expression, and Purification of Dvl1_PDZ Domain

Dvl1_PDZ domain-expressing cells were grown in Luria- Bertani medium at 37°C until they reached an OD600 of 0.7, at which time 0.5 mM isopropyl β-D-thiogalactopyranoside was added, and the cells were cultured for a further 15 h at 18°C. The cells were harvested by centrifugation and stored at -80°C. The harvested cells were disrupted by sonication in lysis buffer [25 mM sodium phosphate, 100 mM NaCl, 5 mM β-mercaptoethanol, and protease inhibitor cocktail (Sigma- Aldrich, St. Louis, MO, USA), pH 7.8]. The hexahistidine- tagged fusion protein was purified by immobilized metal affinity chromatography on an Ni-NTA column (Amersham Pharmacia Biotech, Little Chalfont, UK), and the hexahistidine tag was cleaved by incubation with tobacco etch virus (TEV) protease for 12 h at 25°C. The purified Dvl1_PDZ was then further purified by size-exclusion chromatography (SEC) using a HiLoad Superdex 75 preparative-grade columns (Amersham Pharmacia Biotech) [23].

Aptamer Screening against Dvl1_PDZ Domain using SELEX

To select Dvl1_PDZ domain-specific aptamers, SELEX was performed. Briefly, an ssDNA library template consisting of a 30-nucleotide random region (N30) was prepared. The random regions were flanked by 15-mer primer regions for polymerase chain reaction (PCR) with the following sequence: 5′-GAGTGACCGTCTGCCTG-N30-CAGCCACACCACCAGCC-3′. The ssDNA library was incubated with His-tagged Dvl1_ PDZ domain protein in selection buffer [40 mM HEPES (pH 7.5), 102 mM NaCl, 5 mM KCl, 5 mM MgCl2, and 0.05% Tween-20]. After ssDNA binding, Dvl1_PDZ domain protein was partitioned by immobilizing them on Dynabeads TALON (Invitrogen, Carlsbad, CA, USA), and unbound ssDNAs were removed by washing with selection buffer. ssDNAs were extracted and amplified by conventional PCR using a 5′-OH terminal biotinylated reverse primer. A primer extension was then performed, and an enriched pool was prepared for the next round. After eight rounds of SELEX, the enriched ssDNA pool was cloned and sequenced.

SEC

Purified Dvl1_PDZ domain samples were loaded onto a HiLoad 16/60 Superdex 75 (GE Life Science) gel filtration (AKTA prime) column equilibrated with the same final protein buffer [10 mM HEPES (pH 7.8) and 10 mM NaCl]. The SEC result was monitored by absorbance at 280 nm. The molecular weight of the purified protein was determined with reference to the standard proteins of Dvl1_PDZ domain (11 kDa) and WD-Aptamer (10 kDa). The molecular weight of Dvl1_PDZ domain was calculated by the following equation: (log_Y_ = −1.2483_x_ + 6.2868; R_2 = 0.9943, _x = elution volume).

Fluorescence Spectroscopy

The binding constants between Dvl1_PDZ domain and aptamer were measured by an LS55 fluorescence spectrophotometer (Perkin-Elmer). Dvl1_PDZ domain protein in a buffer containing20 mM Tris (pH 8.0) and 50 mM NaCl was titrated with aptamer to a molar ratio of 1:0 to 1:1 using a thermostat cuvette. The values detected at the same concentration of ligand only were subtracted for the calibration of the inherent intensities of the protein ligands. The dissociation constant (K_d) values were calculated using the following equation: log (_F_0 − _F/F) = log (1/K_d) + _n_log [ligand], where _F_0 and _F are the fluorescence intensities of the protein at 360 nm in the absence and presence of ligand, respectively, and n represents the ligand-binding site of the protein.

Cell penetration and Viability test

HaCaT cells (human skin keratinocyte cell line) were cultured in Dulbecco’s modified Eagle medium supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ mL penicillin, and 100 µg/mL streptomycin (Gibco BRL, Grand Island, NY, USA). The cells were maintained in an incubator (95% air/5% CO2) at 37°C. Cells were seeded in 24-well plates at a density of 1 × 104 cells per well and allowed to grow for 24 h to 60% to 70% confluence. Biotin- labeled aptamers (WD-Aptamer or negative control) were added to the culture medium at a final concentration of 100 nM and incubated with live cells at 37°C for 30 min. After incubation, the cells were fixed with 4% paraformaldehyde at room temperature for 10 min. After fixation, cells were treated with 0.03% Triton X-100 (Sigma-Aldrich) in 1× phosphate-buffered saline and incubated with Alexa Fluor 555 streptavidin (Invitrogen) at room temperature for 30 min in the dark. Finally, the cells were counterstained with 4,6′-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA), and images were obtained by a fluorescence microscope (Optinity, Korea).

Cell viability was performed via 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich). Cells were seeded in 96-well plates at a density of 5 × 104 cells per well and incubated at 37°C for 24 h. After preincubation, cells were treated with various concentrations (0.001, 0.01, 0.1, 1, 10, and 100 µM) of WD- Aptamer for 24 h. MTT solution (final concentration of 0.5 mg/mL) was added to each well, and the cells were incubated at 37°C for 2 h. The supernatant medium was removed, and 100 μL DMSO (Daejung, Korea) was added to each well to extract the insoluble purple formazan crystals. Absorbance was quantified at 570 nm using a microplate reader (Synergy HT; Bio-Tek, USA).

Purification of Dvl1_PDZ Domain and WD- Aptamer Selection

Dvl1_PDZ domain was amplified by PCR. The amplified cDNA fragments were subcloned into the modified expression vector pET21b (Novagen, Madison, WI, USA) with hexahistidine tag and a TEV protease cleavage site at the N terminus. The recombinant plasmid was transformed into Escherichia coli (strain BL21 DE3). The construct of Dvl1_PDZ domain was made, and the overexpressed protein was purified. To identify the purification of target proteins, samples of each step were loaded on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Figure 1a).

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(a)

(b) Figure 1: Purification of Dvl1_PDZ domain and WD-Aptamer selection. (a) Recombinant pET-21b (+) vector map for the expression of Dvl1_PDZ domain of mouse Dvl1. The map of E. coli expression vector containing His-tag, TEV protease cleavage site, and recombinant Dvl1 PDZ is presented as well as the purification result of SDS-PAGE. (b) General scheme of SELEX using an ssDNA library template.

To develop WD-Aptamer, an ssDNA library template consisting of N30 was used to proceed with SELEX (Figure 1b). The output produced by SELEX produced 353,514 sequences via next-generation sequencing. Ten high-scoring candidate sequences were selected.

Interaction Study of Dvl1_PDZ Domain and WD- Aptamer

The SEC binding assay of WD-Aptamer and Dvl1_PDZ domain was studied. The experimental results show that the size of WD-Aptamer + Dvl1_PDZ domain was 20 kDa, whereas that without WD-Aptamer was 9 kDa (Figure 2a). This result provides strong evidence that WD-Aptamer and Dvl1_PDZ domain interact. A study on the interaction of WD-Aptamer with other Wnt/β-catenin signaling component proteins was conducted to confirm that WD-Aptamer specifically binds to Dvl1_PDZ domain (Figure 2b). To establish the direct interaction between WD-Aptamer and Dvl1_PDZ domain by a fluorescence assay (_K_d = 284.8 nM), fluorescence signals acquired by titration between WD-Aptamer and Dvl1_PDZ domain are shown in Figure 2c.

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Figure 2: Interaction study of Dvl1_PDZ domain and WD-Aptamer. SEC binding assay of WD-Aptamer and (a) Dvl1_PDZ domain, (b) Axin_DIX domain, and Dvl1_DAX domain. Both proteins are Wnt/β-catenin signaling component proteins. (c) Fluorescence spectroscopy analyses of the interaction between WD-Aptamer and Dvl1_PDZ domain. The molar ratios of Dvl1_PDZ domain and WD-Aptamer are presented. A.U., absorbance units.

Cell Penetration and Proliferation

To demonstrate the ability of WD-Aptamer to enter HaCaT cells, Alexa Fluor 555-labeled streptavidin was coupled with negative control or WD-Aptamer-biotin under identical conditions to measure fluorescence imaging. The fluorescence signal shows that a large portion of WD- Aptamer was entered into HaCaT cells compared to the negative control (Figure 3a). To examine the cell viability of WD-Aptamer itself, human keratinocyte cells (HaCaT) that spontaneously immortalized human epithelial cell lines were used. This experiment demonstrated that treatment with various concentrations of WD-Aptamer for 24 h significantly increased cell proliferation of human skin keratinocytes in a dose-dependent manner (Figure 3b).

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Figure 3: Cell penetration and proliferation. (a) Biotin-labeled aptamer (red) and nuclei marker DAPI (blue) were incubated with culture cells followed by fixation and imaging by a fluorescence microscope. Scale bar, 100 μm. (b) Cell viability in human keratinocyte cells (HaCaT) was determined using an MTT assay after treatment with WD-Aptamer for 24 h.