The 76nt random oligonucleotide library, 5'-ATC CAG AGT GAC GCA GCA-40nt-TGG ACA CGG TGG CTT AGT-3',FITC labelled upstream primer 5'FITC-ATC CAG AGT GAC GCA GCA, and Biotin labelled downstream primer 5'Biotin-ACT AAG CCA CCG TGT CCA-3' were synthesised by Huada Biotech (Beijing, China) Co., Ltd. U2-OS cells, HOS cells, and HT-1080 cells were purchased from Boshide Bioengineering Co., Ltd. McCoy’s 5A medium and high-sugar DMEM medium were all purchased from Boshide Bioengineering Co., Ltd (Wuhan, China). Yeast tRNA (Fluka Analytical, USA), heat-inactivated foetal bovine serum (FBS Invitrogen, USA), Streptavidin Sepharose® HP (GE healthcare, USA), RPMI-1640 medium (GIBCO), 2x Taq PCR Master Mix, 50 bp DNA Ladder (Tiangen, Japan), and an agarose gel recovery kit (Dingguo, China) were also used.
Cell culture and sub-library construction
McCoy’s 5A and high-sugar DMEM containing 10% foetal bovine serum were employed to culture U2-OS, HOS and HT-1080 tumour cells respectively. Cells with an activity of more than 95% were prepared 24 hours before the experiment, and the cell count was 0.5 × 106 cells/(100 mm × 20 mm) in a Petri dish (cultured overnight). The initial oligonucleotide library 5'-ATC CAG AGT GAC GCA GCA-40nt-TGG ACA CGG TGG CTT AGT-3' was dissolved in 1000 µl of binding buffer (the final concentration of ssDNA was 10 nM for the first binding reaction and 1000 nM for the second binding reaction), denatured at 95 ℃ for 5 minutes, and placed in ice water for later use.
After the cells were thrice-washed with rinsing buffer, the residual liquid was drained, and the precooled binding buffer containing the candidate library was added to the cell culture plate for binding reaction at 4°C and 50 rpm. It took one hour for one to four rounds, and the reaction time was shortened to 30 minutes after four rounds. After the reaction was terminated, the cells were washed with rinsing buffer to remove ssDNAs that did not bind to the cells. Cells were harvested with a cell shovel into a 1.5-ml centrifuge tube, heated at 95°C for 10 min to separate ssDNAs bound to the cell surface from the cells, centrifuged at 13,000 rpm at 4°C for 5 min, and the supernatant was collected. SsDNAs in the supernatant were used as a template, double-stranded oligonucleotides (dsDNAs) were prepared in large quantities by PCR. Streptavidin Sepharose affinity chromatography columns were used to dissociate double strands, and the harvested positive-stranded ssDNAs would be used as an aptamer sub-library for the next round of screening.
Optimisation of template quantitative concentration and amplification cycle times
Each round of ssDNAs dissociated from the cell surface needs PCR amplification to generate enough dsDNA to obtain enough ssDNAs for the next round of aptamer screening. The optimisation of PCR amplification system and parameters is a necessary step. Previous literature reported that the template concentration in the PCR reaction system was basically optimised by percent volume (Su et al. 2018; Tan et al. 2016). For example, in a 100-µl PCR reaction system, 50 µl of 2x Taq PCR Master Mix was included, the final concentration of upstream and downstream primers was 0.5 µM, 10 µl %, 20 µl %, and 40 µl % of template ssDNAs were respectively placed in three reaction tubes, and ddH2O supplemented the reaction volume to 100 µL. In this study, quantitative template concentration was adopted to optimise the cycle times simultaneously: U2-OS ssDNA concentration was measured at 139ng/µl, and 27.8 ng/µl: 13.9 ng/µl and 6.95 ng/µl were obtained after 5-fold, 10-fold, and 20-fold dilution. The 12.5-µl amplification system contained 6.25 µl of 2x Taq PCR Master Mix, 0.5 µl of upstream primer, and 0.5 µl of downstream primer (the final concentration was 0.5 µM), 3 µl of each concentration template, and 2.25 µl of ddH2O. PCR temperature parameters: denaturation temperature 95 ℃ for 30 s, annealing temperature 56.3 ℃, followed by extension at 72 ℃ for 30 s. PCR systems with each template concentration were subjected to 8, 10, 12, and 14 cycles respectively. The results showed that the 6.95 ng/µl × 3 µl template and the bands obtained in 14 cycles were bright and non-specific amplifications 3% agarose gel electrophoresis. The template quantitative experiment of HOS cells was similar to that U2-OS. Whereas, PCR in volume template often exhibited unsatisfactory optimisation results, requiring redesign of template volume, cycle number, and even ion and temperature parameters.
Comparison on affinity monitoring of sub-libraries by fluorescence spectroscopy and flow cytometry
Starting from the sixth round, the screened sub-library ssDNAs were analysed by flow cytometry and fluorescence spectrum images, and subsequent experiments such as whether the screening process was terminated or not, and continuing cloning and sequencing, were determined according to image drift. For flow cytometry monitoring: 5'-FITC-ssDNA was synthesised by the company, or 5'-FITC-forward primer and 5'-Biotin-reverse primer were used to prepare double-labelled products by PCR, and then 5'-FITC-ssDNAs were prepared through alkaline lysis affinity chromatographic column separation. 5'-FITC-ssDNAs obtained by alkaline lysis needed desalting and purification for later use. The reaction process was as follows: 5 µl of 10-µM 5'-FITC-ssDNAs were dissolved in 95 µl binding buffer containing 10% bovine serum albumin (the final concentration was 500 nM), meanwhile, 500 nM unlabelled ssDNA was prepared for blank control. Each target cell used in the experiment was rinsed three times with 0.5 ×106 rinse buffer and dissolved in 100 µl binding buffer (containing 10% FBS); 50 µl of cells and 50 µl of standby ssDNA were gently mixed, and the binding reaction was conducted at 4°C and 50 rpm for 30 minutes. After rinsing the cells, they were dissolved in 200 µl of binding buffer for flow cytometry analysis.
Fluorescence spectrum analysis: the target cells were cultured by counting 0.5 × 106 cells from 24 hours ago, rinsing three times with the rinsing solution in the next day, then the cells were dissolved in the binding buffer solution containing the final concentration of ssDNAs of 1000 nM, and the cells were rinsed after binding reaction at 4°C and 50 rpm for 30 minutes to remove unbound ssDNAs. The cells were then dissolved in 300 µl of binding buffer at 95°C for 10 minutes: ssDNAs bound to the cell membrane surface were separated from the cells, centrifuged at 13,000 rpm for 5 minutes, and the supernatant harvested. 150 µl of the supernatant was used as a template, and PCR amplification was performed using double labelled primers. 200 µl of Streptavidin Sepharose was loaded into the chromatography column, DPBS fully rinsed gel particles in the column, then 300 µl of double-labelled dsDNA from PCR amplification was added, and the chromatography column was fully rinsed with DPBS with at least three column bed volumes. 200 mM NaOH (500 µl) was added for alkaline lysis, and alkaline lysis solution containing 5'FITC-ssDNA-3' of the positive chain was collected. Fluorescence spectrum was used to monitor the lysis solution at an excitation wavelength of 492 nm and emission wavelength of 520 nm.
Reverse screening of sub-libraries of U2-OS and HOS Cells by HT-1080 Cells
Clinical diagnosis of OS needs to be differentiated from fibrosarcoma and chondrosarcoma. Although their affinity has not yet been revealed, the characteristics of inducement, infiltration and metastasis imply that there are similarities between cell epitopes of OS cells and fibrosarcoma cells, so fibrosarcoma cells were selected as reverse screening cells. The reverse screening process was slightly different from the forward screening of target cells: each round of forward screening sub-library ssDNAs was dissolved in 700 µl to 1000 µl binding buffer (final concentration 1000 nM), heated to 95°C for denaturation for 5 minutes, and then placed in ice water for later use. High-sugar DMEM containing 10% FBS cultured HT-1080 tumour cells was used: cells with an activity of more than 95% were prepared 24 hours before the experiment, and the cell count was 0.5 × 106 cells / (100 mm × 20 mm Petri dish) (cultured overnight). After the cells were thrice-washed with rinsing buffer, the residual liquid was drained, and the precooled binding buffer containing library ssDNAs was added to the cell culture plate, and the binding reaction was conducted for 45 min at 4°C and 50 rpm. SsDNAs that had not been bound to HT-1080 cells remained in the binding buffer, so after the reaction was terminated, the liquid in the Petri dish was harvested into a 1.5-ml centrifuge tube, and the OD value was determined. DsDNAs and Streptavidin Sepharose affinity chromatographic columns were prepared in large quantities by PCR according to the above method to dissociate double strands, and the harvested positive strand ssDNAs were purified, whereafter specimens were stored or used for the next round of screening of positive aptamers.