Selection and preliminary application of DNA aptamer targeting A549 excreta in cell culture media

Abstract Lung cancer is one of the malignant tumors which is still threating to human health. Early lung cancer generally shows no obvious symptoms, one of the diagnostic methods of lung cancer is the detection of tumor markers. However, the specificity and detection rate of the existing lung cancer diagnostic markers are rather low, discovering new lung cancer markers is an urgent need for clinical diagnosis. The method of systematic evolution of ligands by exponential enrichment (SELEX) was used for screening aptamers recognizing A549 excreta in cell culture media in a single-stranded DNA library using magnetic beads as supporter, the cell culture media as target and Real-time Quantitative PCR as monitor system. Our study is the first time to apply the method of screening tumor secretory components by using the culture medium with tumor cells as the screening target which is less interfered by other components, so the probability of the screened aptamers targeting tumor excreta with high accuracy is much higher than other methods. Using A549 cell culture media as positive target and the HMVEC cell culture media as negative control target, nine rounds of selection were conducted and some specific aptamers were gradually enriched. After further high-throughput sequencing, artificial selection and structure prediction, we screened seven aptamers as candidate targeting the excreta of A549 cells. Among them, the aptamer Cell APT-2 and Cell APT-4 showed high affinity and strong selectivity to A549 cell culture media with an equilibrium dissociation constant (Kd) of 8.723 ± 2.180 nM and 7.079 ± 1.336 nM respectively. Moreover, the aptamer Cell APT-2 exhibits strong selectivity to lung cancer serum and the aptamer Cell APT-4 showed strong selectivity to A549 cell culture media. In order to further clarify the specificity of aptamers targeted to lung cancer serum, we used 10 lung cancer patients' serum, 10 normal human serum and 10 colorectal cancer patients' serum for specific verification. The results showed that the detection rate of serum with lung cancer was higher than 80%. Therefore, the aptamer Cell APT-2 probably can be used as a probe to catch specific lung cancer secretory protein for the discovery of new tumor markers.