Screening Aptamers Against NGAL With SELEX And Its Preliminary Application

Objective:NGAL,a biomarker for early AKI diagnosis and prediction,also plays multiple roles in tumor progress.Aptamers are a group of DNA/RNA oligonucleotides which can bind to specific targets with high affinity and specificity,thereby providing potential avenues for the diagnosis and therapy for multiple diseases.In this study,we firstly aimed to screen out aptamers against NGAL using the magnetic bead SELEX.Secondly,we hopefully established ELAA detection method based on the specific aptamers described above.And finally,the roles of aptamers in tumor proliferation in vitro were explored,which may provide a novel strategy for anti-tumor drugs discovery.Methods:(1)The specific aptamers against NGAL were screened using the magnetic bead SELEX,and the sequence homology analysis and secondary structure prediction for these aptamers were analyzed,subsequently,the specificity and affinity of candidates were verified with quantitative real-time PCR.(2)A53,the best aptamers among candidates,was used to establish aptamer-antibody-based sandwich-type(ELAA)detection method.Then,the performance of this method was evaluated,including specificity,linear range,sensitivity,precision and accuracy.(3)Immunofluorescence was performed to verify whether A53 can bind to NGAL or not.Clone formation and CCK-8 proliferation assay were both performed to study the roles of A53 on the proliferation of KYSE450 cells,a cell line of esophageal squamous cell carcinoma.Results:(1)After 8 rounds of magnetic beads SELEX and qPCR validation,aptamers A3 6,A42 and A53 were obtained,and they can specifically bind to NGAL with Kd of 43.59nM,66.55nM and 32.52nM,respectively.(2)The results of ELAA performance verification showed that the linear range was 125 ng/mL-4000 ng/mL,the limit of detection was 30.45 ng/mL,and coefficient of variance(CV)was lower than 15%,and recovery was from 80%-110%.(3)The results of Immunofluorescence showed that A53 can bind to NGAL with high specificity.And clone formation and CCK-8 proliferation assay both demonstrated the proliferation of KYSE450 was significantly inhibited after the involvement of FAM-A53 in the medium as compared to control(P<0.05).Conclusion:(1)Three aptamers,A36,A42 and A53,are obtained after SELEX,and they can bind to NGAL with high affinity and specificity.(2)The ELAA detection method with aptamers against NGAL can be potentially applied to the clinical experiment.(3)Aptamers A53 can bind to NGAL with high specificity and inhibit the proliferation of KYSE450 cells.