The Aptamers Selection Of AMPAR

Stroke leads to cell damage which ultimately cause death or disability in human and currently its one of the three major diseases in the world.One reason of Tissue/Cell damaging is Ischemic stroke where the blood supply is low or lack to the brain which cause neuronal diseases.GluR1 abundantly present in brain which play key role in major brain function like learning,memory and cognition by participating in LTP.In addition,GluR1 is associated with a variety of neurological diseases such as Parkinson's syndrome,Huntington and schizophrenia.Our previous data indicates that GluR1 is involved in the process of cerebral ischemia and exhibits dynamic changes therefore GluR1 was thought to be a potential source of drug target for the treatment of a variety of neurological and psychiatric disorders.However,the changes in GluR1 expression at the synaptic level cannot be observed clearly after cerebral ischemia stroke so for that purpose probe was needed to be find which give us clear vision in dynamic changes of GluR1 protein.Aptamers are synthetic,functional single-stranded DNAs that are screened by Exponentially Enriched Ligand Evolution Technology(SELEX).Aptamers has the binding ability from small molecules to cells and bind in a unique three-dimensional structure.Aptamers are ideal tool for bioimaging,clinical diagnosis and treatment.Modified aptamers can penetrate the blood-brain barrier and show great pot ential in neuroscience fields such as drug delivery,neurological diagnosis and treatme nt.We construct GluR1 and GluR2 recombinant vectors and with the use of PEI we transfer the vector into Hela cells.The expression level and localization of GluR1 and GluR2 were identified by using Western Blot and immunofluorescence.Furthermore,we construct a retroviral recombinant vector of GluR1 and GluR2 and after virus packaging the vector was transferred to Hela cells and then construct a stable cell line for expressing AMPAR.The expression and interaction of GluR1 and GluR2 proteins was identified by immunofluorescence and CO-IP.Then we performed cell-based aptamer screening(Cell-SELEX)in these two different modes of expressing AMPAR.The screened library was incubated with target cells to monitor fluorescence enrichment.The results showed that the fluorescence of the library after screening was not enhanced compared to the original fluorescent library as the number of rounds was selected.