Study On The Selection, Identification And Activity Of Aptamers Against Four Organophosphorus Pesticides

Organophosphorus pesticides are commonly applied in agricultural practice in China, and the maximum residues limit is0.2mg/kg in farm products according to Chinese National Standards (GB). And their unreasonable use has seriously endangered Chinese food safety in recent years. Therefore, there is an urgent need to develop fast detection techniques of pesticide residues. Aptamers, as new biological recognition element with potential substitute for antibody, are research focus recently. Aptamers have been isolated against different molecule classes including antibiotic, heavy metal ions, foodborne pathogens, biotoxin and others in food security fields, and various aptamer-based detection assays have been developed for them. However, there is little information about aptamers against organophosphorus pesticides. In this study, DNA aptamers which bind to four organophosphorus pesticides including phorate, profenofos, isocarbophos and omethoate were screened by using the systematic evolution of ligands by exponential enrichment (SELEX). Furthermore, the affinity, specificity and active sites of selected aptamers were studied.The contents and results are as follows:1. Preparation of single-stranded DNA (ssDNA) in screening aptamer by SELEXExperimental conditions of ssDNA preparation by gel capture were studied to lay a foundation for the construction of secondary ssDNA library in screening aptamer by SELEX. The double stranded DNA (dsDNA) labeled with biotin and fluorescence by the polymerase chain reaction were denatured with NaOH after combined with gel labeled with streptavidin. The results showed that the optimized binding time of dsDNA and gel was40minutes, the concentration of NaOH was0.1mol/L. And agarose gel and polyacrylamide gel electrophoresis revealed that the product prepared under optimized condition was ssDNA. The method was also proved to be easy, convenient and to save time in operation. Furthermore, the ssDNAs prepared by the method were labeled with fluorescence, which was easy to monitor the aptamer selection. And it was appropriate for secondary ssDNA library construction when screening aptamer by SELEX.2. Selection of DNA aptamers which bind to four organophosphorus pesticidesDNA aptamers against four organophosphorus pesticides (phorate, profenofos, isocarbophos and omethoate) were simultaneously isolated from an immobilized random ssDNA library by SELEX technique. During each round of selection, the ssDNAs binding to the organophosphorus pesticides were eluted from the immobilized ssDNA library, and enriched as the selection round progressed. At the twelfth selection round, the output/input ratio was40.8%and peaked. The eluted ssDNAs of the twelfth selection were amplified and cloned into a pUC-T simple vector. Fifteen positive colonies with plasmids containing inserted ssDNA were sequenced. Top5ssDNAs with the highest homogeneity in sequence were selected for further characterization. First structures and second structures of5ssDNAs were analysised, which showed that they were homologous, and typically assembled by loop motifs. The loop motifs may be the binding basis between aptamer and four organophosphorus pesticides.3. Methods for activity identification of aptamersThe two fluorescence detection methods based on structure-switching signal and molecular beacon were developed and applied to the activity identification of the selected ssDNAs. The results showed that the reagents background, free quenching sequence Q-B, four organophosphorus pesticides had very little effect on fluorescence intensity with the method based on structure-switching signal. This result demonstrated the method was feasible, and the fluorescence could be better quenched when the ratio of fluorescent sequence F-P1to quenching sequence Q-B was1:2. The experimental results based on molecular beacon indicated that the molecular beacon was rational and stable, whose hairpin could successfully be both closed and opened up. The optimized condition that organophosphorus pesticides and the molecular beacon competitively bound the aptamers was as follows:the ratio of aptamers to molecular beacon was1:1.25, incubated time was50min at room temperature, and the organophosphorus pesticides shoud be added prior to the molecular beacon. The two methods were applied to the activity identification of the5ssDNAs mixture. A comparison between the two methods indicated the method based on structure-switching signal was not very satisfaying because of small change and low recovery of fluorescence intensity. So the method based on molecular beacon was very appropriate for the activity identification of aptamer. 4. Activity and specificity identification of aptamersTo determine the activity of the five selected ssDNAs, binding assay was performed by the competitive inhibition method. In binding to the four target molecules, SS2-55and SS4-54displayed the highest activity in binding to isocarbophos with inhibition ratios of over75%, and their activity in binding to the other three target molecules was also sufficiently high with the inhibition ratios of above39%. The specificity of SS2-55and SS4-54aptamers to nine structurally similar pesticides was also examined. The inhibition ratios of the nine pesticides were below15%, and SS2-55and SS4-54displayed weaker affinity with the above nine pesticides. And the equilibrium dissociation constants of them to organophosphorus pesticides were further tested by a fluorescent combining equilibrium filtration method. The dissociation constants of SS2-55and SS4-54binding to the four organophosphorus pesticides ranged from0.83to2.5μmol/L. These results showed SS2-55and SS4-54aptamers could bind to phorate, profenofos, isocarbophos and omethoate with higher activity and specificity, and they might be broad-spectrum aptamers.5. Study and analysis of aptamers active sitesActive sites of SS2-55and SS4-54aptamers were studied based on structural modification by aptamer cutting and splicing. Activity of modified aptamers was tested by the competitive inhibition method based on molecular beacon. Active sites could be inferred according to the activity change of modified aptamers. And binding sites of Loops were also studied based on the interaction between four organophosphorus pesticides and four bases by ultraviolet absorption spectrum.The results based on aptamer cutting showed that as for SS2-55the Loop2-4was a mutual active site for four organophosphorus pesticides, especially for omethoate it was very important; the Loop2-3was an important active site for phorate; Loop2-1and Loop2-2were mutual active sites for profenofos and isocarbophos. As for SS4-54, the nucleotides at3’and5’ends were important active site for phorate and omethoate; Loop4-2and Loop4-1were mutual active sites for profenofos and isocarbophos; Loop4-3was a mutual active site for profenofos and omethoate. The results based on aptamer segments splicing showed that the binding between aptamers and four organophosphorus pesticides was not a simple stack of active sites, some parts were not binding sites but they were also important for keeping activity of aptamers. In addition, if several bases of aptamers were different, their activity would be of great difference. The results showed four organophosphorus pesticides had effect on ultraviolet absorption spectrum of four bases in varying degrees, the absorption peaks showed red shift and hypochromic effect. As four four organophosphorus pesticides, the interaction between isocarbophos and T, C, A were strongest, then the interaction between omethoate and G, A, C, but phorate and profenofos only had a weak interaction with four bases. T C on active Loops were important binding sites for profenofos, A、G were important binding sites for omethoate, G were important binding sites for profenofos, but the binding between phorate and aptamers cloud not depend on bases.