Screening,Characterization,and Preliminary Application Of Aptamers For α-Conotoxin MI And GI

Conotoxins（CTXs）are mixed polypeptide toxins secreted by cone snails.They can act on a variety of ion channels or neurotransmitter receptors,and cause numbness,convulsions,vomiting,respiratory failure,and other toxic symptoms.There are more than one million kinds of CTXs,among whichα-conotoxin MI（CTX-MI）,α-conotoxin-GI（CTX-GI）,and other highly toxic species are deadly to human beings.Some of them can be used as probes for neurobiological studies because of the specific recognition of targets.In addition,some species such asω-CTXs have pharmaceutical activity and are known as“the treasury of marine drugs”.However,only about 1%of them have been identified,and there are few reports on the mechanism and pharmacological activity.The method of quantitative detection only relies on mass spectrometry.More importantly,the lack of targeted detoxification agents has made CTXs a hidden danger for underwater operators and impeded the promotion of relevant research and the application of CTXs.One of the obstacles to resolving this problem is the absence of specific recognit ion elements.Antibodies are most commonly used as effective recognition elements.But CTXs are haptens,which cannot obtain monoclonal antibodies directly by immunizing animals.Besides,the affinities of recombinant antibodies are difficult to guarantee.Therefore,it is necessary to search for alternative recognition elements for CTXs recognition,enrichment,and clearance.Aptamers have shown high affinities for varieties of targets,and shown great advantages in the specific recognition of small molecular compounds.They are widely used in detection,disease diagnosis,drug development,and other fields.Hence,aptamers are expected to replace antibodies for the recognition of CTXs.In this paper,CTX-MI and CTX-GI,which are the most toxic species,were taken as examples.MB-SELEX was used to obtain the specific aptamers（MBMI-01 and MBGI-02）for CTX-MI and CTX-GI.In addition,MnO₂-SELEX was created for the screening of broad-spectrum aptamers,and Mn-13 was obtained with double target affinity.Then the aforesaid aptamers were truncated based on the secondary structure model and the three-dimensional interaction model,and the active sites of the aptamer were obtained by improved computer simulation.The truncation and mutation of nucleotide sequences were further carried out,which significantly improved the affinity and specificity of aptamers.Finally,based on the optimized aptamers,the biosensor was developed and preliminatively applied to the rapid quantification of CTX-MI and CTX-GI in water and blood samples.The specific results were as follows:1.The aptamers were screened by MB-SELEX and MnO₂-SELEX.MB-SELEX was used for specific aptamers screening.CTX-MI and CTX-GI were respectively used as targets for independent screening.The results of high-throughput sequencing showed that after 12 rounds of positive selection and 6 rounds of counter selection,the diversity of sequences decreased significantly while the high-affinitive sequences showed great enrichment.In addition,MnO₂ nanosheets were used as ss DNA fixation materials and MnO₂-SELEX was designed based on the method of library fixation.The aptamers with broad affinity were obtained after 10 rounds of selection.According to the degree of enrichment and Gibbs free energy,the dominant aptamers were screened,and the affinity was detected by BLI assay.Finally,MBMI-01（K_D=850 n M）,the specific aptamer for CTX-MI,MBGI-02（K_D=610 n M）,the specific aptamer for CTX-GI,and Mn-13（K_D=530and 740 n M with CTX-MI and CTX-GI,respectively）,the broad spectrum aptamer were obtained.2.The post-SELEX process was further introduced to improve the compatibility of aptamers.Firstly,the fixed regions at both ends were removed,and no significant change in affinity was detected in new sequences（MBMI-01R,MBGI-02R,and Mn-13R）.Then,based on the secondary structure model predicted by Mfold,MBGI-02 was truncated to 25nt.The affinity of the optimized aptamer,MBGI-02Rb,was increased from 610 n M to 39n M.However,MBMI-01R and Mn-13R can form G-quadruplets,which have a large deviation from the model predicted by Mfold.The three-dimensional interaction model can directly display the true conformation of the aptamer-target compound,but the existing algorithms cannot predict the interaction between the target and flexible nucleotide chain by simulating nucleotide by nucleotide when the interaction region was unknown.Therefore,taking MBMI-01R as an example,the semi-empirical formula was invoked for algorithm optimization to obtain the three-dimensional interaction model of MBMI-01R and CTX-MI,and the interaction region was determined to be a pocket composed of 11nucleotides residues,in which CTX-MI could be specifically fixed through hydrogen bond network.Then,based on this model,truncation and mutation optimization were performed to increase interaction sites,reduce steric hindrance and simplify the G-quadruplet skeleton.Finally,a 35-nt aptamer,MBMI-01R₆,was obtained,which improved the affinity of CTX-MI from 850 n M to 60.5 n M.3.BLI-based aptasensor were developed by MBMI-01R₆ and MBGI-02Rb.The aptasensors can be stored for a month and reused at least 10 times.The linear detection range of CTX-MI aptasensor was 1-50μM,and the LOD and LOQ were 77.5 and 258.2n M,respectively.The CTX-GI aptasensor had a linear detection range of 2.5-70μM,with LOD of 128.8 n M and LOQ of 429.2 n M.Seawater,tap water,and blood samples were then tested,and the recoveries were 92.22-109.35%,while the coefficient of variation was less than 7.5%.It is proved that these aptasensors can be used for the quantitative detection of CTX-MI and CTX-GI in samples.Compared with the existing methods,the aptasensors can complete a test within 10 minutes without complex sample purification.Additionally,all the consumables can be reused.Hence,these aptasensors are more suitable for local detection,batch screening,and rapid diagnosis.In summary,after screening,characterization,and optimization,the specific and high-affinitive aptamers for CTX-MI and CTX-GI were obtained,and they were preliminatively applied for rapid quantification in samples.The aptasensors are hopeful to replace existing methods for field detection and rapid diagnosis.In addition,the procedural process is also applicable for other CTXs to obtain molecular recognition elements.