Study On The Screening And Application Of N-acetylneuraminic Acid-specific Single-stranded DNA Aptamers

Sialic acid(SA),also known as neuraminic acid,is acylated derivatives of a hydroxylated monosaccharide containing 9 carbon atoms.The most common SA is N-acetylneuraminic acid(Neu5Ac),which plays important roles in cell migration,infant development,and functional plasticity of the nervous system.Meanwhile,sialylation helps tumor cells to proliferate,metastasize and evade recognition by the immune system.Therefore,the detection of Neu5Ac not only provides guide to evaluate normal and pathological cellular processes,but also is helpful in analysis of functional contents in food.The main research contents and results are as follows:(1)DNA library-immobilized magnetic beads(MB)-based SELEX and two different target-immobilized MB-based SELEX were used to screen the Neu5Ac-specific aptamers from the 79 nt-length single-stranded DNA(ss DNA)initial library with 35 nt random sequences in the middle.After multiple rounds of screening,the affinity of the enriched sequences for Neu5Ac reached the maximum.After that,the pooled products from the final round of screening were PCR amplified,gel recovered,cloned and sequenced.(2)RNA Structure software was used to simulate the secondary structure of each screened sequence.After that,the sequences were divided into different families,and the optimal sequences were selected from different families to measure the affinity.Through the comparison of the measured data,the optimal sequences from the three methods are ap 1-5,ap 2-33,and ap 3-1,and their dissociation constants(K_d)are 126.85±11.40 nmol·L^-1,132.32±28.60 nmol·L^-1,87.34±15.19 nmol·L^-1,respectively.(3)The screened aptamers,ap 1-5,ap 2-33 and ap 3-1 were subjected for molecular docking simulation and binding site prediction.The candidate sequence was optimized according to the secondary structure and molecular simulation docking.By removing the non-binding site fragment from the sequence,the length of the truncated aptamer ap 3-1-a is only 21 nt.The K_dvalue of ap 3-1-a is 55.71±12.29 nmol·L^-1,indicating that the affinity is36.21%higher than that of ap 3-1 before truncation.Thus ap 3-1-a was determined to be the optimal aptamer of Neu5Ac.According to the process parameters of SELEX and the performance of the obtained aptamers,DNA library-immobilized MB-SELEX technique is determined to be the most effective screening method for small molecule targets.(4)To verify the practicability of the aptamer,a fluorescent biosensor for Neu5Ac was constructed based on the adsorption and fluorescence quenching of 6-FAM-labeled ap 3-1-a by graphene oxide(GO)and subsequent fluorescence recovery by Neu5Ac.Neu5Ac was detected under the optimal conditions.The results show that the fluorescence intensity increases with the increase of Neu5Ac concentration.When the concentration of Neu5Ac is in the range of 20-1000 nmol·L^-1,the fluorescence intensity shows a linear relationship with the concentration of Neu5Ac.When the actual samples were tested,the recovery rate of the method was ranged from 90.60-101.80%with the relative standard deviation(RSD)of2.80-5.63%.The specificity of the biosensor was verified.The fluorescence response after the addition of Neu5Ac was significantly stronger than those of the structural analogues of the target,while the fluorescence signal generated by the other carbohydrate compounds could be ignored.The aptamer obtained through screening and the established fluorescence detection method have achieved rapid,simple and more sensitive detection of Neu5Ac,and has a good application prospect in clinical diagnosis and disease treatment.