Selection,Identification And In Vivo Imaging Study Of Endoglin Specific Aptamers

Background:Tumor vascular endothelical cells are essential to tumor angiogenesis,which play a very important role in tumor growth and metastasis.Early diagnosis and treatment against tumor vascular endothelial have become a major trend in cancer studies.Endoglin is a transmembrane glycoprotein which is expressed on the surface of tumor endothelia cells.As it's overexpressed in proliferative endothelia cells,but not expressed or only weakly expressed in normal endothelial cells,Endoglin is recognized as a specific marker for tumor angiogenesis and neovascularization.Aptamers are a class of DNA or RNA sequences which could bind to their target with high affinity and specificity.Aptamers have a similar effect with antibodies and often referred as "Chemical antibodies".In recent years,with the development of aptamers selection technology,more and more aptamers for tumor cells were selected and observed.However,these tumor-associated aptamers could recognize only a kind of tumor cells,which are useless in other cancers.It largely limits the application of aptamers.Endoglin is a perfect target for aptamer selection because of general and broad expression in vascular endothelia cells in solid tumors.Nevertheless,there is still no report of aptamers against Endoglin.Objective:In vitro selection and biological effects detection of DNA aptamers specifically against Endoglin molecule to obtain DNA aptamers which can be used in vivo against tumor tissue.We suggest a new method which has potential applications for the targeted diagnosis and treatment of tumor targeting.Methods:According to Cell-SELEX method,we synthesized a random oligonucleotide library with 84bp sequences(containing a 45 random bases sequence).Then,we constructed a stable cell line which expressed human Endoglin protein on 293T cells(293T-hE cells)stably,and used it as the target cell.We chose 293T cells as negative control cells in selection.The selection pressure is gradually strengthened to improve selection efficiency.When the selection library was enriched,we sent the enriched DNA library for TA cloning and sequencing.The results of cloning and sequencing was analyzed and compared according to their primary and secondary structure.Next,we selected the representative candidate aptamers of each DNA family and synthesized FITC-labled for flow cytometry assay and fluorescence imaging.Finally,the candidate aptamers with the best binding ability were truncated and modified with CY5 to investigate their binding affinity in vivo to tumor tissue.Results:1.After 19 rounds selection,we obtained five candidate aptamers which could bind to 293T-hE cells named ENG-1,ENG-2,ENG-3,ENG-4 and ENG-5.The flow cytometry assay showed good Kd values in low nanomolar level,wherein the ENG-4 had a good affinity with only a 5.58±3.11 nM Kd value.2.These five candidate aptamers were detected at different temperatures in 4 ? and 37 ?.All of the five aptamers could bind to Endoglin expressed cells,confirmed these aptamers have a good biological effect,not affected by environmental temperature.3.We truncated and modified the sequence of highest affinity,aptamer ENG-4.In our test,we found a new aptamer which has a similar binding ability with ENG-4 named ENG-4c.In the subsequent sections of tumor tissue in vivo imaging assay,its excellent targeting ability was also confirmed.Conclusion:In this study,we selected and observed aptamers which can bind to Endoglin molecule with high specificity and affinity.The aptamers showed great potential for the application in tumor targeted diagnosis and treatment.