Genomic DNA of EN2 was purchased from Thermo Fisher Scientific (USA). The pET28a plasmid was purchased from Novagen (Germany). Nucleospin Gel and PCR Clean-up/Plasmid EasyPure kit were acquired from Macherey-Nagel (Germany). E. coli strain Rosetta (DEC), Dynabeads his-tag Isolation and Pulldown, MyOne Streptavidin C1, and TOPO TA cloning kit were purchased from Invitrogen (USA). HisTrap Ni-NTA affinity column and Sephadex G-25 Superfine resin were purchased from GE Healthcare (USA). All DNAs were synthesized from Bionics Inc. (South Korea). For ELONA, EN2 monoclonal antibody was purchased from Abnova (Taiwan). Streptavidin Poly-HRP (poly-HRP), 3,3′,5,5′-tetramethylbenzidine (TMB) substrate kit, 96-well polystyrene plates, and sulfuric acid (H2SO4) were purchased form Thermo Fisher Scientific (USA). Bovine serum albumin (BSA) and IgG were purchased from Sigma-Aldrich (USA). Prostate specific antigen (PSA) and cancer antigen 125 (CA125) were purchased from Sino Biological Inc. (USA) and Abnova (Taiwan). Artificial urine medium (AUM) was purchased form Pickering Laboratories Inc. (USA). All materials were of analytical grade. All aqueous solutions were prepared in deionized water (> 18 MΩ) obtained using a Direct-Q system from Merck Millipore (USA).
Preparation and purification of EN2
The EN2 genes, which corresponds to protein P19622 (UniProtKB database), were amplified using PCR with sequence specific-forward primers including BamHⅠ linker (EFP, 5’-CCC GGA TCC ATG GAG GAG AAT GAC CCC AAG C-3’) and reverse primers including XhoⅠ linker (ERP, 5’-CCC CTC GAG CTA CTC GCT GTC CGA CTT GC-3’). PCR products were purified, and subsequently digested using BamHⅠ and XhoⅠ. They were cloned into pET28a vectors that had been pre-digested under the same conditions, which leads to the addition of the His-tag at the N-terminal of EN2. The plasmids were then transformed into E. coli Rosetta (DEC) to allow over-expression of EN2.
Bacteria were grown at 37℃ in lysogeny broth (LB) supplemented with 50 µg/mL kanamycin and 25 µg/mL chloramphenicol until the optical density (OD) at 600 nm reached 0.6. Point expression was induced by addition of 200 µM isopropyl β-d-1-thiogalactopyranoside (IPTG) and incubation at 37℃ for an additional 6 h.
Bacterial cells were harvested and sonicated, then the lysate was cleared by centrifugation at 18,000 rpm for 40 min and applied to a HisTrap Ni-NTA column. The fusion proteins were eluted with an imidazole gradient, then the eluates were added to a desalting column with storage buffer (50 mM Tris-HCl, 100 mM NaCl, 0.5 mM β-mercaptoethanol, and 5% (v/v) glycerol, pH 8.0). The products were stored at -80℃ in aliquots containing 20% (v/v) glycerol, then analyzed using 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
in vitro selection for EN2-specific ssDNA aptamer
The overall in vitro selection was conducted using magnetic bead SELEX and performed in 100 µL of binding buffer (20 mM Tris–HCl, 50 mM NaCl, 5 mM KCl, 5 mM MgCl2, pH 8.0)27,28. A library template was synthesized as ssDNA containing a central random region of 40 nucleotides (DNA library: 5’-CAC CTA ATA CGA CTC ACT ATA GCG GAT CCG A-N40-CTG GCT CGA ACA AGC TTG C-3’). For the selection, 20 µL of pre-washed NTA magnetic beads (Dynabeads His-Tag Isolation & Pulldown) were incubated with 500 pmol of His-tagged EN2 for 1 h at room temperature (RT), then washed to remove unbound proteins using an external magnetic separator. Then 500 pmol of ssDNA library was heated at 95℃ for 5 min then cooled on ice for 1 h to stabilize the naturally-occurring secondary structures, then incubated with EN2-immobilized magnetic beads for 1 h at RT. Unbound ssDNAs were collected and measured by UV absorbance at 260 nm to calculate the amount of bound ssDNAs.
Then the EN2-ssDNA complexes were eluted using binding buffer supplemented with 300 mM imidazole, and the eluted ssDNAs were precipitated using 70% (v/v) ethanol and amplified by PCR with pfu polymerase, using forward primers (5’-CAC CTA ATA CGA CTC ACT ATA GCG GA-3’) and biotinylated reverse primers (5’-biotin-GCA AGC TTG TTC GAG CCA G-3’). The resulting dsDNAs were added to 70 µL of streptavidin-coated magnetic beads (Dynabeads MyOne Streptavidin C1) in coupling buffer (5 mM Tris-HCl, 1 M NaCl, 0.5 mM EDTA, 0.0025% (v/v) tween-20, pH 7.5) for 1 h at RT, then washed in coupling buffer. Then non-biotinylated ssDNAs were eluted using 200 mM NaOH and sequentially precipitated using 70% (v/v) ethanol. The generated ssDNAs were used as the library for the next round of SELEX. After the 12th round of SELEX, the eluted ssDNAs from the EN2-immobilized magnetic beads were amplified by PCR using unmodified primers.
Finally, the amplified dsDNAs were cloned into pCR 2.1-TOPO TA vectors, and the constructs were transformed to E. coli TOP10 cells (TOPO TA Cloning Kit). The plasmids were purified using a Nucleospin Plasmid EasyPure kit, and the inserts were sequenced. The secondary structures of the aptamer candidates were predicted using the Mfold program (http://www.unafold.org/mfold/applications/dna-folding-form.php)29.
Measurement of dissociation constant ( K d )
The dissociation constants Kd were determined using a magnetic-bead fluorescent assay. 5’ FAM modified aptamers were heated at 95℃ for 5 min then cooled on ice for 1 h in binding buffer prior to use. First, 100 pmol of His-tagged EN2 was shake-incubated with 10 µL of pre-washed NTA magnetic beads for 1 h at RT, then the EN2-immobilized beads were separated using an external magnetic separator. Next, the EN2-immobilized beads were reacted with several concentrations (0–100 nM) of 5’-FAM modified aptamers for 1 h at RT in darkness. The suspensions were washed twice to remove unbound aptamers, then the EN2-aptamer complexes were obtained using binding buffer supplemented with 300 mM imidazole. Finally, the fluorescence intensities of the eluates were measured by 485/20 nm excitation and 528/20 nm emission filter on a Synergy™ HT microplate reader (BioTek, USA). Kd values were calculated by fitting the data to the one-site-binding (total and nonspecific binding) model of GraphPad Prism 5.0 software (GraphPad, USA)30.
Aptamer-antibody hybrid ELONA that uses HCR
In preparation, 5’biotinylated detector, hairpin 1 (H1), and hairpin 2 (H2) were heated at 95°C for 5 min, then cooled on ice for 30 min in binding buffer. First, 96-well polystyrene plates were coated with 5 µg/mL of EN2 monoclonal antibody (100 µL) at 4℃ overnight. The plates were blocked using 200 µL of blocking buffer (10 mg/mL BSA in PBS) for 1 h at RT. Subsequently, the plates were washed with wash buffer (0.05% (v/v) tween-20 in PBS), then a two-fold serial dilution of EN2 (0, 0.20, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 100 and 200 nM) in PBS was added individually to wells of the plates for 1 h at RT. At the same time, detector, H1, and H2 were mixed to 100 nM each, then reacted for 1 h at RT to complete the HCR process. Then the plates were washed three times with wash buffer, and the HCR products were treated for 1 h at RT. The plates were washed three times, then incubated with 100 µL of poly-HRP diluted 1:500 in blocking buffer for 5 min at RT. The plates were washed five times, then reacted with TMB solution for 20 min at RT in darkness. The reactions were stopped by 1 M sulfuric acid, and the absorbance at 450 nm (A450) was measured using a Synergy™ HT microplate reader (BioTek, USA). For EN2-spiked urine samples, a two-fold serial dilution of EN2 from 800 nM was prepared in PBS including 50% (v/v) AUM, and other procedures were performed as above. All data were obtained in triplicate at each EN2 concentration. To analyze the sensitivity of the assay, the limit of detection (LOD) was calculated as 3σ/s, where σ is the standard deviation (s.d.) of the sample absorbance, and s is the slope of the linear relationship between A450 and EN2 concentration. The coefficient of variation (CV) was calculated as the ratio of s.d. to the mean.