Rational Truncation Optimization And Binding Of 17β-estradiol Aptamer

Background:17β-estradiolum(E2)is one of endocrine disrupting chemicals(EDCs),which is easy to accumulate through the food chain and is harmful to human body.Therefore,strengthening the monitoring of E2residue level in food and environment is one of the important measures to ensure food and environment safety.In the laboratory,traditional instrumental analysis,such as gas chromatography(GC),high performance liquid chromatography(HPLC),gas chromatography-mass spectrometry(GC-MS)and liquid chromatography-mass spectrometry(LC-MS),has been widely developed.However,due to the complex sample pretreatment process,high cost and technical limitations,the rapid detection of E2 is very difficult.Therefore,it is particularly important to develop a simple,low-cost,high selectivity and high sensitivity E2 detection method.Nucleic acid aptamers have been widely used as recognition elements since their discovery.Minimizing the length of aptamers can not only reduce the synthesis cost,but also make aptamers have higher affinity or better selectivity.Molecular dynamics(MD)simulation is a powerful method to analyze the interaction between aptamers and target molecules,which is conducive to improve the truncation efficiency and success rate of aptamers.Objectives:Based on MD simulation,the reported E2 aptamers were truncated and modified.The interaction between E2 and a series of designed aptamers was explored by computer,and its binding sites were explored to compare the binding affinity.Gold nanoparticles(Au NPs)colorimetry was used to verify the affinity of the truncated aptamer,and the dissociation constant(K_d)was measured to verify the reliability of molecular dynamics simulation results.At the same time,whether the truncated aptamer can be used for E2 detection was explored,and an E2colorimetric sensor based on Au NPs was constructed for E2 detection in food and environment.Methods:(1)Based on the original aptamer,a series of truncated aptamers were designed,and the molecular docking software autodock4.2.6 was used to dock the original aptamer and truncated aptamer with E2,preliminarily explore their binding sites and compare their binding affinity;(2)According to the autodock docking results,MD simulation was carried out with GROMACS 4.5.5,and each conformation with the lowest binding energy was selected as the dominant conformation for simulation research.The dynamic binding process of aptamer and E2 was observed within 100ns,and the binding stability,binding sites and energy were analyzed;(3)The affinity and selectivity of the truncated aptamer were verified by Au NPs colorimetry,and the K_d value was measured.The truncated aptamer with good affinity and selectivity was selected to construct an E2colorimetric sensor based on Au NPs to determine E2 in milk and tap water.Results:(1)Autodock software was used to explore the binding affinity and binding sites of aptamer E01-E13 and E2.The results showed that aptamers E01,E02,E04 and E09-E12 had high affinity for E2 and had more binding sites.(2)The 100 ns MD simulation of E2-aptamer complex system revealed several important characteristics of the aptamer bound to E2:there is a ring structure containing T or G base;The size and structure of the ring would affect the bonding strength;The binding of G base to E2is stronger than that of T base.(3)The affinity of aptamers was compared.The preliminary comparison results were basically consistent with the prediction results of binding free energy in Chapter 2 and Chapter 3.At the same time,the K_d value was measured to verify the specificity of truncated aptamer E09-E12.A colorimetric method based on Au NPs was established to detect E2,with a linear range of 0.25-10μg/m L wide linear range,which was also suitable for the detection of E2 in mineral water and milk.Conclusion:Computer means can effectively predict the affinity of aptamers,simulate the binding process,determine the basic binding sites between aptamers and targets,explore the interaction mechanism between aptamers and targets,and provide effective technical support for the truncation and optimization of aptamers.At the same time,a colorimetric method based on Au NPs was established to detect E2 in the environment.The sample preparation is simple and time-consuming.