Development and Validation of a Novel Quality Control Methodology on Screening Out for Human Osteosarcoma Cell Aptamers

Background: We have successfully established an aptamer library for recognition of human osteosarcoma cell using the cell-SELEX method. However, we found that the quality monitoring become a key, resulting in success or failure in the screening process. The study aims to establish an accurate, time-saving quality-monitoring method in screening for tumour cell aptamers in order to shorten the screening process and ensure the accurate preparation of the aptamer. Methods: Two kinds of human osteosarcoma cells (U2-OS, HOS) were selected as the forward screening target cells and human fibrosarcoma cells (HT-1080) as the reverse screening cells to screen the aptamers from the candidate oligonucleotide library. In each round of preparation of the library, PCR was optimised by using quantitative template concentration instead of percentage volume. Each round of forward screening was conducted with reverse screening; Fluorescence spectroscopy and flow cytometry were used to monitor and compare the aptamer libraries. Results: During quantitative PCR for U2-OS and HOS template, the results showed that the bands obtained from 14 cycles were bright and no non-specific amplification within the optimal template concentrations between 19.0 and 21.0ng/µl. Each round of forward screening was accompanied by reverse screening to accelerate the elimination of non-specific single-strand DNA (ssDNA). In the meanwhile, the aptamer groups were effectively purified specifically bounding to target cells. Besides, we observed that the fluorescence spectroscopy is more accurate, time-saving, and convenient for quality control compared with flow cytometry. Conclusion: The method proposed in the study is appropriate for the rapid screening out for human osteosarcoma cell aptamers. The quantitative template concentration, forward screening with back screening, and fluorescence spectroscopy are important methods for accurate preparation and quality control of tumour cell aptamers. It can provide scientific reference data for the amplification of dsDNAs in other sub-libraries.