The Mechanism Of A DNA Aptamer Targeting Mesenchymal Stromal Cell And Its Application In Monitoring The Homing Of Transplanted MSCs

Due to the lack of tools that can provide accurate information about the migration and survival status of mesenchymal stromal cells（MSCs）in vivo,the main objective of this research is to avoid any modification of MSCs and provide a method that can accurately track the migration and survival of MSCs in the boday.Aptamers undergo significant conformational changes when they bind to their targets,so novel biosensors with various multipurpose properties can be designed on the basis of this feature.And their low cost and mass synthesis properties make them ideal for clinical applications.In the cell-SELEX process,cell surface proteins remain in their natural conformation and maintain their complete biological function,so that the aptamer can fully recognize the molecular characteristics of a specific cell.In our previous study,we employed cell-SELEX technology to obtain the aptamer W2 of m BMSCs using peripheral blood cells as negative cells.Flow cytometry analysis proved that aptamer W2 could bind MSCs with high specificity and high affinity across species（human,rat and mouse）.In addition,the mesenchymal stem cell line can be separate and purify from mixed cells by W2 in vitro.This result indicates that the aptamer W2 has stability and targeting in a complex system,laying the foundation for in vivo applications.Cell-based SELEX can facilitate the discovery of target cell biomarkers,therefore,we explored the target protein of W2 to further understand the mechanism by which W2recognizes MSCs.The target protein was enriched by the W2-mediated affinity pull-down experiment,and the molecular weight of the target protein was analyzed by 2D gel electrophoresis.Then,the binding affinity of the fluorescently labeled W2 to different cells was correlated with the gene expression level of this cells was compared and analyzed.The experimental results showed that fibronectin（FN）may be a potential target protein of W2.Further flow cytometry showed that the binding ability of W2 to FN knockdown MSCs（FN^--MSCs）was reduced,and it was also confirmed by EMSA that the protein interacts with W2 is FN.In order to explore the mechanism of the recognition of FN by aptamer W2,through the electrophoretic mobility shift assay,it was found that W2 recognizes fibronectin as cellular fibronectin（c FN）with an extended structure,without binding to plasma fibronectin（p FN）in compact conformation.Furthermore,it was also found that heparin could inhibit the binding of aptamer W2 to c FN.Therefore,according to the three different haparin binding sites on the single subunit of FN,the m RNA of FN was divided into three segments and expressed on HEK293 cells respectively.Flow cytometry revealed that W2 only binds to HEK293 cells that highly expressed Hep2 domain(FNⅢ_12-14).Moreover,molecular modeling through bioinformatics also proved that the binding site of aptamer W2 and heparin on fibronectin are sterically hindered.To investigate the application of W2 in biomedicine,aptamer W2 was designed as an activatable aptamer-based fluorescence probe（AAFP）for tracking of living cells,and the homing of transplanted m BMSCs in a mouse model of chronic kidney disease was monitored in real time by AAFP.And compared with the widely used magnetic resonance imaging（MRI）based on Fe₃O₄nanoparticles.Both MRI and AAFP-based fluorescence imaging results showed that the infused m BMSCs mainly accumulated in the liver in control mice;however,in the CKD mice,m BMSCs were more inclined to homing to the damaged kidneys and promoted renal repair.However,in histological analysis,it was found that CA@Fe3O4nanoparticles were not only present in the injured glomeruli,but also scattered irregularly in normal tissues,which indicated that there were some false positives in MRI due to the escape of nanoparticles.But the fluorescence signal of AAFP only exists in the glomerulus,indicating that compared with MRI,AAFP had higher accuracy and higher signal-to-noise ratio in identifying m BMSCs.Therefore,AAFP-based fluorescence imaging is superior to MR imaging in revealing the duration and survival status of m BMSCs migration and homing to injured tissue.In conclusion,this study developed an aptamer that can target and recognize MSCs in vivo and in vitro,and this binding event is achieved through the Hep2 binding site on the c Fn protein.The technology of tracking MSCs with W2-based aptamer probe not only circumvents the shortcomings of various modifications or transformations of MSCs in previous studies,but also has advantages over traditional MR imaging technology in showing the survival status of migrated and colonized m BMSCs.Meanwhile,this study not only provides a tool for tracking exogenous MSC in vivo,but also lays a foundation for future research on the biological role and ultimate fate of MSCs in regenerating tissues.