br Fig A Identification of potential aptamer candidates Binding
Fig. 2. (A) Identification of potential aptamer candidates. Binding assays of the selected aptamers, PDGC21 and PDGC45, with the target Oleic acid BGC-823 and the negative cells SGC-7901. (B) The secondary structures of the aptamers PDGC21 and PDGC45 were predicted using the web-based IDT OligoAnalyzer. The structures in red parentheses represent the truncated forms. (C) Bar graph of the flow cytometry assays results for the binding of aptamers PDGC21 and PDGC21-T with BGC-823 cells. * *p < 0.01 (D) BGC-823 and SGC-7901 cells were incubated with FAM-labelled PDGC21-T and FAM-labelled library, respectively. Fluorescent images revealed that PDGC21-T specifically bound to the surface of BGC-823 cells. Scale bar was 20 µm.
We performed the Cell-SELEX at 4 °C to avoid enriching nonspecific DNA sequences, which can occur by endocytosis at 37 °C. Considering that the aptamer acts as a targeted molecular probe and may be used in environments with various temperatures, we further verified the target recognition of PDGC21-T at room temperature (25 °C) and physiolo-gical temperature (37 °C). As shown in Fig. 3B, aptamer PDGC21-T exhibited similar binding to target BGC-823 cells at temperatures of 25 °C, 37 °C and 4 °C, indicating that the ability of aptamer PDGC21-T to bind to the target BGC-823 cells is not affected by temperature. This finding suggests the possibility for aptamer PDGC21-T to be used under varying circumstances.
The target binding experiments confirmed that aptamer PDGC21-T can recognize the poorly differentiated GC cell line BGC-823 but not the moderately differentiated GC cell line SGC-7901 (Fig. 2A). To further verify this differentiation-specific binding, we examined the binding specificity of aptamer PDGC21-T against two other GC cell lines, MGC-803 and MKN28. As shown in Fig. 4, aptamer PDGC21-T only bound to MGC-803 cells but not MKN28 cells, further validating that aptamer PDGC21-T specifically binds poorly differentiated GC cells, as MGC-803 is a poorly differentiated GC cell line and MKN28 is a well-differ-entiated GC cell line [34,35]. Moreover, this phenomenon also occurred in colorectal cancer (CRC) cells; aptamer PDGC21-T was shown to
Fig. 3. Binding properties of aptamer PDGC21-T. (A) Binding curve of aptamer PDGC21-T with BGC-823 cells. Cells were incubated with varying concentrations of FAM-labelled PDGC21-T in triplicate. (B) Flow cytometry assays were performed to assess the binding capacity of PDGC21-T at 4 °C, 25 °C, and 37 °C, respectively. The red curve represents the background binding of the ssDNA library.
recognize the poorly differentiated CRC cell line SW620 but not the better differentiated CRC cell lines HT29 or CCL187 [36,37]. In addi-tion, there was no obvious signal detected when any normal cell lines were tested, such as GES-1, HEK293, NIH3T3, CHO or COS-7, following treatment with FAM-labelled PDGC21-T, implying that the aptamer PDGC21-T possessed not only differentiation specificity but also cancer specificity.
3.4. QD605 labelled aptamer PDGC21-T for targeted imaging using immunohistochemistry
Immunohistochemistry is one of the most commonly applied clinical methods for analysing the molecular and morphologic characteristics of tissues, and antibodies have been used extensively as the molecular probes for immunohistochemistry . However, antibody production
is a time-consuming process that still relies heavily on the use of ani-mals; in addition, inter-batch variation in antibodies can occur . Thus, aptamers, which are known as chemical antibodies, may be an alternative to conventional antibodies, and they show promise in ex-panding the histological staining of tissue sections for the targeted imaging of biomarkers of tumour cells [40–42]. Aptamers are more effective than antibodies, given that aptamers can resist thermal or pH changes, can be easily modified with imaging probes through covalent conjugation, and they have a smaller size, which can improve tissue penetration and increase systemic clearance [43,44]. Shigdar et al. demonstrated that the EpCAM aptamer exhibited superior staining compared with conventional antibodies in paraffin embedded tissues, especially for certain antigens with low expression levels .