Selection And Identification Of Aptamers To Rabbit Mesenchymal Stem Cells

Objective : To evolve the ss DNA aptamers specifically binding to rabbit mesenchymal stem cells(MSCs),for future applications of recognizing MSCs from multiple species origin and sorting different subpopulation of MSCs.Methods:MSCs were isolated from thighbone of immature rabbit and identified by induced osteogenic and adipogenic differentiation respectively.Three types of aptamers to cell surface,cell surface protein and internalization were evolved by cell SELEX(systematic evolution of ligands by exponential enrichment)technique with different ss DNA elution methods from MSCs.After eight rounds of selection,enrichment of the 5th and 8th pools were evaluated through binding assay of FAM modified pool to MSCs by confocal microscopy.The enriched 5th pool targeting cell surface and surface protein respectively and8 th internalized ss DNA were then cloned into p GM-T vectors and cloned sequences were determined randomly.The candidates were chosen based onprimary sequence conservation and predicted secondary structure by RNA structure and MEME online analysis.The candidates were compared by flow cytometry analysis and confocal imaging.Aptamer 5-1-9 with stronger binding ability was chosen for sorting positive cells by flow cytometry for further analysis and characterization of target.Flow cytometry analysis was also used to identify the recognition of candidate aptamers to MSCs of rabbit,rat and human origins.Results : In this study,the isolated MSCs have potentials of osteogenic differentiation and adipogenic differentiation under certain conditions.After eight rounds of selection,the 5th pool to cell surface and surface protein respectively and 8th internalized ss DNA showed enrichment compared to naive library and were cloned into p GM-T vectors for sequencing.Fifteen candidate aptamers(from three groups)were picked out of 51 random sequenced clones based on primary and secondary structures and subjected to binding assays by flow cytometry analysis and confocal imaging.The candidates5-1-9,5-1-12,5-1-19,5-2-4,5-2-9,5-2-13,8-3-2,8-3-15,8-3-19 have stronger binding ability.At the same time,the membrane permeability test showed that FAM-labeled single sequence-bound mesenchymal stem cells were mostly viable cells.Cells sorted by sequence 5-1-9 were verified by confocal with stronger fluorescence intensity.We also showed that several candidate aptamers such as 5-1-9,5-1-12 could not only recognize rabbit MSCs,but also bind to MSCs of rat and human origin.Conclusion : A number of ss DNA aptamers to different cellular components of rabbit MSCs were evolved by cell-SELEX that can recognize MSCs of different species origin.Aptamer 5-1-9 could be used to sort MSCs subpolulation.