br The FAM labelled selected aptamers were
The FAM-labelled selected aptamers were synthesized by Sangon Biotechnology Co., Ltd. (Shanghai, China). To determine the Kd value of the selected aptamers, BGC-823 Pepstatin A were incubated with various concentrations of FAM-labelled aptamers in BB at 4 °C for 30 min, and the fluorescence intensity was then determined by flow cytometry. The ssDNA library was used as the negative control. Then, the equilibrium dissociation constant (Kd) of each aptamer was determined by nonlinear regression of one-site binding, according to the following equation, using Sigma Plot 10.0 software (Jandel Scientific, UK): Y = Bmax × X/ (Kd + X), where Bmax is the saturated binding, Y is the fluorescence intensity and X is the concentration of aptamers. All experiments were repeated three times. To determine the cell specificity of the selected aptamers, cancer cell lines, including MGC-803, MKN28, SW620, HT29, CCL187, and normal cell lines, including GES-1, CHO, COS-7, HEK293, and NIH3T3, were used to analyse the binding ability with flow cytometry, as de-scribed above. All experiments were repeated three times.
2.6. Fluorescence microscopy analysis
The cells were seeded in the chamber and cultured overnight. After being washed twice with WB, the cells were incubated with 250 nM FAM-labelled aptamers or ssDNA library at 4 °C for 1 h. Then, after being washed twice with WB, the cells were fixed with 4% for-maldehyde for 15 min and stained with DAPI for 5 min to counterstain the nucleus. The fluorescence images of the cells were obtained with an Olympus IX51 fluorescence microscope (Olympus, Japan).
Aptamer PDGC21-T is a truncated sequence of aptamer PDGC21. To detect the targeting ability of aptamer PDGC21-T, the non-target cells SGC-7901 were incubated with Cell-tracker Green (Invitrogen) for 30 min at 37 °C and then digested with trypsin and mixed with the BGC-823 cells. For fluorescence imaging, biotin-labelled PDGC21-T was in-cubated with the cells at 4 °C for 1 h. The biotin-labelled PDGC21-T was synthesized by Sangon Biotechnology Co., Ltd. After washing, the cells were incubated with QD605-SA (Wuhan Jiayuan Quantum Dots Co., Ltd., China) at room temperature for 15 min. The fluorescence images of the cells were then obtained.
2.7. Effect of temperature on the binding of BGC-823 cells
analysis. The ssDNA library was used as the negative control. All ex-periments were repeated three times.
2.8. Tissue targeted imaging analysis
The GC TMA was preheated at 60 °C for 4 h and then deparaffinized in xylene (15 min, twice). The TMA was then immersed in decreasing concentrations of ethanol (100%, 95%, 90%, 80% and 70%) at 5 min intervals. The hydrated tissues were pre-treated in 0.01 M citrate buffer (pH 6.0) and heated in a pressure cooker for 100 s. Afterwards, the tissue sections were blocked with pre-cooled BB contain 20% FBS and 20% BSA for 1 h at 4 °C and incubated with biotin-labelled PDGC21-T or the ssDNA library for 1 h at 4 °C. After being washed three times with PBS, the tissue sections were incubated with QD605-SA for 15 min, and the fluorescence signals were examined under a laser scanning confocal microscope (LSM510, Carl Zeiss Meditec AG) with a 488-nm argon laser.
2.9. Statistical analysis
All experimental data were presented as the mean ± standard de-viation. The Student's t-test and one-way analysis of variance were used to analyse the results. The statistical analyses were performed using GraphPad Prism software.
3. Results and discussion
3.1. Selection and enrichment of aptamers specific to poorly differentiated BGC-823 cells
To obtain DNA aptamers specific for poorly differentiated GC, we employed a Cell-SELEX approach using the poorly differentiated GC line BGC-823 as the target cells. Counter-selection with the moderately differentiated GC cell line SGC-7901 was introduced in the 4th round and performed in all subsequent rounds to eliminate the possibility of any sequences recognizing surface molecules common to both the target and negative cell lines. The enrichment of the ssDNA pool through successive selection was monitored by flow cytometry. Gradual shifts in fluorescence intensity were observed in subsequent rounds.
The starting ssDNA library, 3rd round, 6th round, 9th round, 12th round and 15th round pools were tested for their binding affinity for the target BGC-823 and negative SGC-7901 cells. As shown in Figs. 1A and 1B, with the increasing cycles of selection, there was a steady increase in the fluorescence intensity on the target BGC-823 cells, whereas there was no obvious change in fluorescence intensity on the negative SGC-7901 cells, indicating that the enriched ssDNA sequences possessed high binding specificity for the target BGC-823 cells but not for the SGC-7901 cells. However, no further increase was detected from the 12th to 15th round, implying that the enrichment saturation of ssDNA sequences had bound to the BGC-823 cells. The specific binding of the 15th round pool to the BGC-823 cells was further confirmed using fluorescence microscopy (Fig. 1C). Compared to the ssDNA library, a meaningful green fluorescence was observed on the surface of the target BGC-823 cells after the 15th round pool treatment. In contrast, there was no detectable fluorescence on the SGC-7901 cells, further con-firming that the enriched ssDNA sequences specifically bound to the target BGC-823 cells.