The Engineering Of Split Aptamers And Investigation Of Its Interaction With Targets Using Atomic Force Spectroscopy

Split aptamers were obtained by dividing the intact aptamer,which could selfassemble to produce a positive signal when the target was present.Split aptamers have shorter nucleic acid strands,which could avoid the formation of complex secondary structures,and it was conducive to the formation of the target recognition configuration.Simultaneously,they could reduce non-specific signals generated by intramolecular interactions effectively.Therefore,split aptamers have been widely used in biosensing,bioimaging,drug delivery and therapy.Given that split aptamers were still in a minority,and there was an urgent need for reliable methods to obtain split aptamers.Atomic Force Microscope(AFM)could investigate the interaction between biomolecules at the single molecule level,and has been widely used in chemistry,biology and materials.Since its advantages in the investigation of biomolecular interactions,AFM was expected to develop into a powerful tool for engineering split aptamers.Accordingly,the thesis investigated the interaction of split aptamers and vascular epithelial growth factor 165(VEGF165)using AFM at firstly,and AFM was proved to be suitable for investigating the interaction between split aptamers and targets.Then,using myoglobin as a model,AFM was applied to engineer the split aptamers against myoglobin,and the obtained split aptamers was used for targets detection.The content of this thesis was as follows:1.Investigating the interaction between split aptamer and vascular endothelial growth factor 165 using atomic force spectroscopyInvestigating the interaction of split aptamer and proteins at single molecule level could provide more biomolecular information,but so far there have been few reports.Herein,VEGF165,a major biomarker of human diseases,was used as a model,the feasibility of investigating the interaction between split aptamer and proteins with AFM was explored.The split aptamers were modified on the AFM tip and substrate respectively,and the interaction between VEGF165 and its split aptamers were mesured in single molecule level using.This work provided a basis for the engineering of split aptamers.2.Engineering of myoglobin binding split aptamer using atomic force spectroscopyDue to the lack of binding mechanism and reliable methods,there were still few split aptamers.In this work,cardiac biomarker myoglobin was selected as a model,a new strategy of engineering split aptamer was explored with AFM.At first,the intact aptamer against myoglobin was randomly divided at different sites.Then,split aptamers were attached to AFM tip and gold substrate respectively,and AFM measurements were carried out in the absence or presence of myoglobin.Given that there was a significant difference in the binding probability with or without the target,it was speculated that the binding probability may be one of the judgment parameters for engineering split aptamer.Three pairs of split aptamers were successfully acquired based on the differences in binding probability.Due to its intuitiveness and reliability,this method could promote the development of split aptamers,and provide a new platform for engineering of split aptamers.3.Detection of myoglobin with split aptamersThe obtained split aptamers were designed for myoglobin detection based on dynamic light scattering and surface plasmon resonance method.Results found that the S2-3 elicited the highest affinity,and the dynamic light scattering method and surface plasma resonance method based on S2-3 functionized gold nanoparticles could realize the detection of nM-level myoglobin in human serum,which could satisfy the needs of clinical detection.