Study The Interaction Between A549Cell Line And Its Aptamers Using Single Molecular Force Spectroscopy Technology

Aptamers are single-stranded oligonucleotides, which are generated fromcell-based systematic evolution of ligands by exponential enrichment (cell-SELEX).Because of its high specificity and affinity, easy synthesis and modification, aptamershave been paid attention in biology and medical fields. Although it has been knownthat aptamers can fold into distinct tertiary structures and bind their targets viahydrogen bonds, hydrophobic interactions and van der Waals interactions, the bindingmechanism of aptamer to its target was rarely investigated, especially the interactionbetween aptamer and cell. Studying the interaction between aptamer and cell at singlemolecule level would be helpful to clarify the information of binding mechanism,dissociation processes and binding sites on the cell surface. It would also providetheoretical basis for designing aptamer molecular probes, reforming stability andbinding affinity of aptamers, and designing aptamer-based biosensors. Therefore, ithas become a controversy issue in biochemistry and biomedical research.In this thesis, single-molecule force spectroscopy technique was applied to studythe interaction between A549cell and its four aptamers (including aptamer S1,aptamer S11e, aptamer S15and aptamerS6) in the living cells level. The maincontents are as follows:1. The interaction force between A549cell and its four aptamers in the livingcells level was studied by single molecule force spectroscopy (SMFS). Informationthat described the different binding behaviors among these four aptamers wereobtained, including the unbinding force, dissociation rate constant at zero force koffand activation energy. The results revealed that the unbinding process of theaptamer/A549cell complexes all have two activation barriers and one intermediatestate, and the binding capacity of A549cells with its four aptamers were closelyrelated with the activation energy barrier. Furthermore, the binding capacity betweenA549cells and its four aptamers closely related with the activation energy barrier.2. The binding site between A549cell and its four aptamers in the living cellslevel was studied by SMFS. A549cells were first blocked using one kind of aptamers,and then the binding forces of three other aptamers to A549cells were analyzed usingSMFS. The results showed that four kinds of aptamers have different binding sites onthe surface of A549cell. Furthermore, the results were also confirmed by flowcytometry. In conclusion, the studies would provide valuable information to clarify theinteraction mechanism between cell and aptamer.