Structural Optimization, Characterization And Application Of Aflatoxins Aptamers

The known content of mycotoxin binding to aptamers has not yet revealed the molecular interaction mechanism of aptamer binding to the target,which severely limits the development of aptamers.At the same time,the strong specificity of the aptamer also makes the aptamer impeded in the ductility of the function.Therefore,in order to provide theoretical guidance for the design,screening and structure-activity relationship of aptamer molecular probes,this study used the mycotoxin AFB₁ aptamer as the research object.The aptamer sequence structure was optimized by sequence tailoring optimization,base mutation substitution and the like.Combined with isothermal titration calorimetry and fluorescence analysis,the molecular basis of the"aptamer-target"recognition process was deeply studied,and the molecular mechanism of the interaction between the aptamer and the target molecule was revealed.Firstly,we truncated the sequence based on the secondary structure of the original aptamer of AFB₁.The secondary structure of the AFB₁ aptamer was analyzed by software Mfold.Considering the interaction structure and stability of the aptamer,the appropriate truncated optimization was carried out on the basis of retaining the stem-loop structure.The affinity and specificity of the truncated aptamer were verified by ITC method and fluorescence analysis to confirm whether the truncated optimization was effective.Apt-2 is the one with the shortest number of bases in the effective truncated aptamer.Its Kd value,binding site number and thermodynamic parameters are similar to the original aptamer and have no binding effect on other mycotoxins.Therefore,the best aptamer in this truncated optimization experiment is Apt-2.Secondly,we designed a site for base mutation based on the secondary structure of the truncated aptamer Apt-2 obtained above.Affinity verification was performed by the ITC method and the fluorescence analysis method,and it was found that mutation of any"loop"site resulted in loss of aptamer affinity.Therefore,it is speculated that Apt-2 is a binding unit by using the entire"loop"-like part to recognize and capture the target in the environment with high affinity,thereby realizing the binding process.The binding sites of AFB₁ were explored by performing ITC experiments and fluorescence analysis experiments on the simple structural constituents of AFB₁ and the structural analogs of AFB₁.It was found that AFB₂,AFG₁ and AFM₁ were strongly bound to Apt-2 by ITC method and fluorescence analysis.Therefore,the site where AFB₁ and the three structural analogs bind to Apt-2 is a central structural site shared by the four toxins.Therefore,AFB₁ and structural analogs expose the central binding site,which is recognized by the"loop"portion of Apt-2 to further capture binding.Thirdly,we established aptamer affinity column of aflatoxin based on the aptamer Apt-2which has strong affinity for the four aflatoxins AFB₁,AFB₂,AFG₁ and AFM₁.The aptamer affinity column is mainly used for enrichment and purification of aflatoxins in real samples.The streptavidin agarose gel and the biotin-modified aptamer were coupled at a volume ratio of 1:2（aptamer concentration of 2μM）to obtain a conjugate as a carrier,which was packed and assembled into an adapter.The aptamer affinity column can exert maximum efficiency at a pH of about 7.4,a temperature of about 25°C,and an organic content of less than 3%.The target binding rate of the aptamer affinity column can be maintained above 85%at no more than 8 uses,while the aptamer affinity column has a single maximum column capacity of 200ng aflatoxins.For other species of mycotoxins,this aptamer affinity column does not bind and exhibits good specificity.The aptamer affinity column can be used for the interception and enrichment of aflatoxin in the real samples for the purpose of sample purification.Fourth,in order to eliminate the obstacles of aptamer specificity to the study of multifunctional aptamers and based on the principle of ensuring economics,we used a small fragment base sequence as a bridging medium to bridge the aptamer Apt-2 with the aptamer of OTA to obtain a bridged aptamer.The 5’and 3’fluorophores of the bridged aptamer are quenched by the complementary strands resulting in the disappearance of fluorescence.When the bridged aptamer binds to the target and the fluorescence is restored,it is effective to characterize the bridged aptamer.The affinity of the aptamers was verified by ITC experiments on the bridging aptamers.It was found by ITC that QL-A did not have affinity for other mycotoxins.The bridged aptamer QL-A has a lower Kd value than a single aptamer.Therefore,the affinity of the bridged aptamer is enhanced while still maintaining good specificity that does not bind to other mycotoxins.