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    2020-08-12

    r> 3.2. Identification and characterization of selected aptamers
    The 15th pools were PCR-amplified, cloned and sequenced. Fifty clones were randomly selected for the sequencing analysis, and positive results were available from 40 clones. Based on the repeatability of the sequences, sequences PDGC21 and PDGC45 with repetitions of 15 and 8 times were chosen to be synthesized for further characterization (Table S2). To confirm the specific binding affinity of the two selected
    Fig. 1. The binding affinity assays of the enriched pools with cells. (A) Flow cytometry assay to monitor the binding of selected pools (3rd, 6th, 9th, 12th, and 15th) with BGC-823 (target cells) and SGC-7901 (negative cells). (B) Bar graph of the flow cytometry assay results. (C) The fluorescence imaging of the 15th pools bound to BGC-823 and SGC-7901 Pepstatin A using fluorescence microscopy. In each picture, left is the fluorescence image and right is the optical image. Scale bar was 50 µm.
    sequences to BGC-823 cells, we labelled the sequences with FAM at the 5′ end and then subjected the cells to flow cytometry. As shown in Fig. 2A, compared to the ssDNA library, PDGC21 and PDGC45 both bound to BGC-823 cells, resulting in an obvious fluorescence shift, but these sequences did not bind to SGC-7901 cells, suggesting that the binding of these two sequences to the target cells is specific. Previous studies have revealed that short, low molecular weight aptamers reduce the cost of chemical synthesis and increase the struc-tural stability and tissue penetration [29]; in addition, it has been re-ported that not all the nucleotides within the aptamer sequences are responsible for the targeted binding [30]. Thus, the full-length apta-mers PDGC21 and PDGC45 were further truncated by removing nu-cleotides at the 5′ and 3′ termini. First, we predicted the secondary structure of aptamers PDGC21 and PDGC45 using the web-based IDT OligoAnalyzer 3.1 programme, which is based on the free energy minimization algorithm. According to the prediction, PDGC21 has two loop structures and PDGC45 has three ones (Fig. 2B). In general, the stem-loop structures of aptamers are thought to play an important role as binding motifs in target recognition [31]. Accordingly, we truncated PDGC21 and PDGC45 by retaining loop related sequences and re-moving some nucleotides in the 5′ end and 3′ end (see the red box section in Fig. 2B, Table S2 and S3). Next, we evaluated their binding affinity with BGC-823 and SGC-7901 cells by flow cytometry. Fig. 2C shows that the truncated aptamers PDGC21-T and PDGC45-T main-tained their binding specificity with the targeted BGC-823 cells but did not bind to SGC-7901 cells; moreover, PDGC21-T exhibited enhanced binding to the target BGC-823 cells compared with the full length ap-tamer, PDGC21, whereas PDGC45-T exhibited a binding affinity equal 
    to that of the full aptamer, PDGC45. The binding of aptamer PDGC21-T to BGC-823 cells was further confirmed by fluorescence microscopy. As shown in Fig. 2D, there was no obvious fluorescence on BGC-823 or SGC-7901 cells following treatment with the ssDNA library; however, intense fluorescence was observed on the surface of BGC-823 cells, but not SGC-7901 cells, following treatment with aptamer PDGC21-T. This finding further indicated that the truncated aptamer PDGC21-T could selectively bind to BGC-823 cells.
    3.3. Characterization of the properties of PDGC21-T for targeted imaging
    To investigate the ability of PDGC21-T to serve as an imaging probe, we analysed its binding affinity, binding selectivity and temperature adaptability. Based on the higher affinity of aptamers to target cells compared with antibodies, aptamer-based targeted molecular imaging has enormous potential application in preclinical cancer diagnostics [32,33]. For example, Shi et al. reported an aptamer for tumour ima-ging in mice that could effectively bind tumours with high affinity, thereby establishing the efficacy of fluorescent molecular aptamer for diagnostic application [32]. To determine the binding affinity of the selected aptamers, the mean fluorescence intensity of various con-centrations of the aptamers during binding assays with BGC-823 cells was measured to determine the dissociation constant (Kd) by flow cy-tometry. Fig. 3A and Fig. S1 show the binding saturation curves of aptamers PDGC21-T and PDGC45-T with target BGC-823 cells. Aptamer PDGC21-T exhibited a better binding affinity, with a K d value of 35.2 ± 1.1 nM, so we chose aptamer PDGC21-T for further evaluation for targeted imaging.
    Fig. 2. (A) Identification of potential aptamer candidates. Binding assays of the selected aptamers, PDGC21 and PDGC45, with the target cells 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.