Over-Expression of CD20 in HEK293T Cells The CD20-encoding pcDNA3.1/Hygro(+) plasmid (pcDNA-CD20) was used to stably transfect the Human embryonic kidney cells 293 (HEK293T) using Lipofectamine 2000. and AP-3. The AP-1 aptamer was the most thermodynamically stable one (GAP-1 = ?10.87 kcal/mol) with the highest binding affinity to CD20 (96.91 4.5 nM). Since, the CD20 is a suitable target for recognition of B-Cell. The selected aptamers could be comparable to antibodies with many advantages. The AP-1, AP-2 and AP-3 could be candidate instead of antibodies for diagnostic and therapeutic applications in immune deficiency, autoimmune diseases, leukemia and lymphoma. Keywords: ssDNA aptamer, CD20, Cell-SELEX, HEK293T cells CDKN2B 1. Introduction Aptamers are biomolecular ligands composed of single-stranded (ss) nucleic acid (DNA or RNA) molecules that are specifically isolated from a random sequence pool via in vitro selection process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX) [1,2]. These specific molecules are able to target or bind a wide range of specific ligands, metal ions, small molecules, ITE proteins, cell surface antigens and whole cells or tissues [3,4]. ITE On the one hand, aptamers have multiple advantages over antibodies such as smaller size; easy, quick, and reproducible synthesis, with low batch-to-batch variability during mass production; low immunogenicity in the body; low toxicity; and modification flexibility. On the other hand, it is indeed possible to select an aptamer molecule with a unique conformation and high binding affinity, with high specificity to a target molecule [4,5]. Because of these features, the aptamers can be considered as an alternative of antibodies [6]. Further, these characteristics render them ideal probes for the clinical research, diagnostics and for therapeutic applications [6,7]. The B-lymphocyte antigen (CD20) protein is a non-glycosylated phosphoprotein encoded by a member of the membrane-spanning 4A gene family (MS4A) and exposed on the surface of the B-cells [8,9]. This tetra-span membrane antigen-like protein is expressed on precursor and mature B lymphocytes, from the early pre-B to the late B stage, except in normal plasma cells and pro-B cells [10]. The membrane-attached CD20 protein functions as a membrane ion channel specifically, a Ca2+ channel that regulates Ca2+ concentration following B-cell antigen receptor (BCR) induction, and authorizes the activation of B-cells [11,12]. The protein is involved in B-cell growth, proliferation, and cell cycle signaling by triggering intracellular tyrosine kinase signaling pathways. These functions depend on the various expression of CD20, e.g., the manifestation of CD20 on B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is definitely 30C50%; and in mature B-ALL or Burkitt-type leukemia/lymphoma, it is 80C90%. This trend is used to facilitate analysis and prognosis of different types of B-cell acute lymphoblastic leukemia (B-ALL) [13,14]. For instance, anti-CD20 monoclonal antibody enables the immunophenotyping of B-cell lineages [14]. Along with getting more knowledge about the part of CD20, antibodies against CD20 have been recently developed to treat numerous B-cell malignancies [15]. Rituximab is an Food and Drug Administration (FDA) authorized anti-CD20 chimeric monoclonal antibody and offers significantly improved the outcomes of the Burkitt-type ALL, chronic lymphocytic leukemia (CLL), and Non-Hodgkins lymphoma treatments [16]. Monoclonal antibody therapy might constitute an optional approach for the treatment of disorders associated with autoantibody production and immune-mediated ailments, such as rheumatoid arthritis, systemic lupus erythematosus, pemphigus foliaceus, dermatomyositis, and idiopathic thrombocytopenic purpura, as well as post-transplant lymphoproliferative disorders (PTLD) [14,17]. Furthermore, monoclonal antibody therapy directed against the CD20 antigen does not target plasma cells, and consequently, serum immunoglobulins generating and immunological memory space cells [14,18]. There is no competition between plasma and lymphoma cell antigens for binding to rituximab because CD20 antigen is not released from the surface of B-cells and, hence, free CD20 antigen is generally not present in the serum in the soluble form [19]. Therefore, based on these special roles, CD20 ITE antigen is an important target for the analysis and treatment of B cell related disease such as autoimmune diseases and B-ALL [2,20]. The sequence of CD20 protein exposed the extracellular website of CD20 consists of loops located near the cell surface. This would enable targeting of the CD20 membrane protein by aptamers, which are similar with monoclonal antibodies [11,12]. Though, monoclonal antibodies take action differently against each of the CD20 epitopes and there is also a great desire for the production of novel antibodies against CD20, but due to some of the disadvantages of classical antibodies such as thermal instability and immunogenicity, this makes limitation of using antibodies and leading ITE to create alternative molecules resemble to them. Among these alternate molecules, the production of aptamers were regarded as [21]. Aptamers are desired because of quick detection methods, cheap, fast and reliable with no difficulty. On the other hand, the cell-selected aptamers against CD20 can provide distinguishable tools in diagnostics and therapeutics. Therefore the design, selection and production of them were desired. In the current study, ssDNA aptamers directed against HEK293T cells expressing CD20 were designed and selected via Cell-SELEX and finally their.