Selection, Binding Mechanism, And Application Of Aptamers Against Gonyautoxin 1/4

Gonyautoxin 1/4(GTX 1/4)is one of the most representative neurotoxins in paralytic shellfish toxins,which is synthesized by marine algae,can be accumulated in fish or shellfish through the aquatic food chain.The ingestion of GTX 1/4-contaminated seafood by human causes paralytic shellfish poisoning characterized by symptoms such as muscle weakness,headache,nausea,dyskinesia,breathing difficulties,or even suffocation.The toxicity of GTX 1/4 is assumed to be due to the inhibition of Na+ channels on the nerve cells and the subsequent blockage of neuronal transmission.At present,the commonly used methods for marine biotoxins detection include mouse bioassay,liquid chromatography and immunoassay method.Since they are time-consuming with low sensitivity and poor stability,these detection methods can not meet the demand for rapid detection of marine biotoxins.Therefore,there is still an urgent need to develop a sensitive and specific new detection method,which can perform rapid quantitative analysis for the marine neurotoxin GTX 1/4.Aptamers,as a new molecular recognition element,has been combined with the biosensor platform and developed into a variety of novel detection methods,which has been widely applied in the analysis,diagnosis,food safety and other fields.In this study,two different SELEX experiments(GO-SELEX and MB-SELEX)were conducted to select DNA aptamers that bind with high affinity and specificity to marine biotoxin GTX 1/4.In the process of selection,we found that GO-SELEX,as a novel screening technology,has significant advantages compared to typical MB-SELEX and is also a simplified,high-speed and immobilization-free method for small-molecule DNA aptamer development.Furthermore,we truncated GTX1/4 aptamer based on the structural prediction of the mfold web server and obtained the aptamer core sequence with a higher Kd of 17.7 nM,indicating that the methodology for the structural prediction of aptamers can be applied to study the relationship between structure and function and to optimize aptamers.To deeply study the binding mechanism between GO18-T-d and target molecule GTX 1/4,we designed a series of molecular modeling,docking and molecular dynamics simulation.Results showed that aptamer GO18-T-d formed an anti-parallel G-quadruplex structure and GTX 1/4 could combine steadily in the groove at the top of the G-quadruplex structure,ledding to high affinity and specificity.Biolayer interferometry(BLI)technology is a highly sensitive,label-free and real-time sensor platform.When combined with aptamer to construct new detection methods,it has good application prospects.Therefore,the aptamer GO18-T-d was used as a recognition molecular element and combined with BLI to construct a sensitive and specific aptasensor for rapid detectionof GTX 1/4.The aptasensor exhibits a broad detection range(0.2 to 200 ng/mL)and a good linearity(0.2 to 90 ng/mL),with a low detection limit of 50 pg/mL.Moreover,it has no cross reactivity with GTX 2/3,STX and neo STX,which are structural analogues of GTX 1/4.Meanwhile,the aptasensor has a high recovery percentage(86.70-101.29%)and low coefficient of variation(0.35 to 6.19%)in real samples.Therefore,this study established an aptasensor with high sensitivity,specificity,stability,immobilization-free and other advantages.We believe that this novel BLI aptasensor offers a promising alternative to traditional analytical methods for rapid detection analysis and on-site real-time monitoring of marine biotoxin GTX 1/4.