Chromosome conformation capture
The juxtaposed MYC/TRD fragment in the SKW3 cell line was analyzed based on our recent study to design suitable detection and capture probes. As we mentioned in our previous study, 3C Library preparation was operated on SKW3 and HUVEC cells. These cells were suspended in the 3C buffer [25] and fixative reagent (Formaldehyde 37%). DNA digestion was done by EcoRI (Jena Bioscience, Germany) according to the manufacturer’s protocol. Proteinase K and its buffer were added to the 3C library to reverse the cross-linked DNA. After phenol-chloroform purification, the SKW3 cell line 3C library was analyzed through agarose gel electrophoresis [26]. For inverse PCR, two regions were selected upstream and downstream of the viewpoint locus on the MYC gene based on EcoRI restriction sites, as shown in Figure 6a. PCR products were sequenced using standard Sanger sequencing service (Bioneer Company, South Korea). The juxtaposed fragment sequence was used for confirming translocation PCR using two specific primers.
DNA-DNA hybridization assay
As described above, 184 bp sequence from translocation PCR was selected for screening probe synthesis. These probes contain 27 bp fragments on the MYC gene (MYC-AuNP-probe) and the TRD gene (TRD-probe).
AuNps synthesis
The most common method for preparing colloidal gold involves reducing gold (III) ions by citrate ions in a dilute aqueous solution (10 mM). Gold (III) ions are usually added to water in hydrogen tetrachloride (HAuCl4). Citrate ions (C6H5O7 -3) are more likely to be oxidized to acetone d-carboxylate ions (C5H4O4 -2), acting as a two-electron reducing agent (Figure 6b).
The reaction for the synthesis of colloidal gold is given in Equations 1 and 2 [27]:
1. Au+3(aq) + 3e- → Au(s)
2. C6H5O7 -3 (aq) + H2O(l) → C5H4O4 -2 (aq) + CO2(g) + H3O + (aq) + 2e
Spectral information
Colloidal gold contains AuNps that measure about 5 to 50 nm. An absorption peak at 520 nm indicates the formation of AuNps with a 15-40 nm diameter. This optical property of AuNps is unique in their commercial products such as medical diagnostic kits, biosensors, DNA analysis, lasers, and optical filters. To prepare a concentration of 10 mM, dissolve one g of gold powder in 250 ml of distilled water containing a dilute solution of 10 mM citrate. After observing the purple color and taking an absorption peak at 520 nm, we brought it to a concentration of 0.5 mM. Also, the size distribution diagram of synthesized NPs with dynamic light scattering (DLS) was investigated.
Conjugation of AuNps and MYC-thiol-modified -probe
To conjugate a thiol-modified lyophilized MYC-probe and a colloidal AuNps, it is necessary to reduce the thiol unit to connect with the AuNps. For this purpose, a 0.1 M TCEP solution was prepared fresh as required and was added directly to the lyophilized probes. To reduction of the thiol group, the tube was incubated at room temperature for one hour. During the incubation time, the sample vortexed and spined several times. Then three M sodium acetate with pH = 5.2 was added, and the tube was vortexed. Subsequently, 1.5 ml of absolute ethanol was added and stored at -20 ° C for 30 minutes, and the tube was centrifuged at 12,000 rpm for 10 minutes.
The precipitated probe was dissolved in sterile DNase-free water after drying (at concentration 1µg/µl). In a sterile tube, nine μl of the reduced probe and five ml of AuNps were mixed and kept at room temperature for 16 hours without shaking. Then solvent buffer No. one, including 0.15 M NaCl, tween 20 0.025%, Tris-HCl 10 mM (PH = 7), and EDTA 2.5 mM (PH = 8) up to a volume of 50 ml was added to them. After incubation at room temperature for 40 hours, the mixture was centrifuged (10,000 rpm, 10 min).
Subsequently, the pellet was dissolved in solvent buffer No. two containing 0.15 M NaCl, tween 20 0.025%, Tris-HCl 10 mM (PH = 7) and EDTA 2 mM (PH = 8). The concentration of this oligo attached to the NPs was measured and used for the colorimetric assay. We named this detection probe MYC-AuNP-probe. Also, to ensure a proper functionalization of AuNps with oligo, their absorption was measured at a 400-600 nm wavelength.
Hybridization assay
Hybridization was performed in 6 groups with the same concentrations of TRD-probe, different concentrations of MYC-AuNp-probe, and 3C-libraries. All steps are the same for different groups as follows:
The nitrocellulose membrane was cut by punching into circular pieces with a diameter of 6.4 mm. These circles are the attachment surface for capture probes and the 3C libraries. These membranes were soaked in SSC (saline-sodium citrate) 20X buffer for 30 minutes before TRD-probes immobilization. Next, the desired values of TRD-probes were placed at 100 °C for 10 minutes and then five minutes on ice to ensure that all probe parts were denatured and single-stranded. Then two µg of TRD-probe was dotted without contact of the tip on the membrane. Then, membranes were placed at 80 °C for two hours and transferred to a 96 well plate to immobilize the probe. Next, the pre-hybridization step was performed by pouring 200 µl of a pre-hybridization buffer. This buffer contains 6X SSC, 4X Denhardt's [9], 10 µl Salmon Sperm DNA, and 0.2% SDS for 4 hours at 65 °C. Buffer was removed, and a 200 μl hybridization buffer containing the same materials and values from the 3C library samples of SKW3 and HUVEC cells was replaced in separate wells. The 3C libraries must be denatured (100 °C for 10 minutes and then freeze for 5 minutes) before adding to the hybridization buffer. For the hybridization step, a library sample and a probe attached to the membrane were incubated for 24-28 hours at 65 °C. Washing was performed by pouring 100 μl of SSC 4X (wash buffer) and then 100 μl of SSC 0.4X at 65 °C for 15 minutes to remove non-hybridized DNA fragments from the reaction.
After removing the wash buffer, different concentrations of conjugated AuNps of 100 ng / µl MYC-AuNp-probe (one μg of MYC-AuNp-probe) and 100 μl SSC 4X were added to each well. For hybridization of the conjugated probe with AuNps, a temperature of 65 degrees and six to 18 hours were applied. After the hybridization step, the remaining nanoparticle solution was removed without contact of the tip with the membrane. Washing consisted of three steps pouring 100 μl of SSC 4X, then 100 μl of SSC 0.4X at 65 °C for 15 minutes, and finally PBS at room temperature, respectively. The colored dots were photographed immediately after drying, and color intensity was analyzed by Image J software version 1.52.
Quantification of the colorimetric assay
Image J software was used to calculate and compare the color intensity in normal (HUVEC) and cancerous (SKW3) samples. A histogram tool was used to calculate the color intensity. The higher color intensity is related to a smaller number in a histogram, and the lower color intensity is related to alarger number and the closer it is to 255 (Figure 4a).
Statistical analysis
The paired t-test was used to compare groups. Data analysis was carried out by GraphPad Prism version 8. Results are reported as the mean ± SD and P < 0.05 were considered to be significant.