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Screening of high-affinity DNA aptamers with SELEX as efficient detection probes for TNF-α Speaker: Wan-Ting Hsu Advisor: Min-Jen Tseng Date: 2010.06.11 Tumor necrosis factor (TNF-) is the prototypic ligand of the TNF superfamily and a mediator of inflammation and immunity. It is secreted by various cells including adipocytes, activated monocytes, macrophages, B cells, T cells and fibroblasts. TNF-α is primarily produced as a transmembrance protein arranged in stable homotrimers. From this membrane-integrated form the soluble homotrimeric cytokine (sTNF) is a 17.5 kDa, 157 amino acid protein released via proteolytic cleavage by the TACE. TNF- signals through two receptors, TNFR1 and TNFR2. Upon contact with their ligand, that can activate NF-B, MAPK pathways, or death signal. TNF promotes the inflammatory response, which, in turn, causes many of the clinical problems associated with autoimmune disorders such as rheumatoid arthritis and ankylosing spondylisis. Nowadays, the most widely detection technique in TNF- is enzyme-linked immunosorbent assay (ELISA). Though ELISA is easy to detect, but this technique is not specific and hard to scale-up for rapid and large quantity sample. Recent researches reveal aptamers are generally discovered using the in vitro selection process referred to as SELEX (Systematic Evolution of Ligands by EXponential enrichment). SELEX is a screening technique used to identify a highly specific aptamer with high binding affinity to a chosen molecular target from a large pool of nucleic acids. The aim of this study is to apply SELEX to screen DNA aptamers with high-specificity and high-affinity to sTNF- protein for developing a quick, specific detective and quantitative method. The cDNA of sTNF-α protein was subcloned into pGEX-KG for overexpression of GST-TNF-α fusion protein. The purified GST-TNF-α fusion proteins were bound to MagneGST glutathione particles for specific DNA aptapmer selection from the 64 mer or 84 mer random ssDNA library with SEXLX. Via several cycles of SELEX selection, DNA aptamers were amplified by PCR, then cloned into yT&A and sequenced. The secondary structures of 67 sequences of aptamers were predicted using Mfold software, two kinds of secondary structure motif were found. To enhance the aptamers specificity, more selection cycles will be performed to identify conserve sequences in the structure. The candidate aptamers will be synthesized for chemical and physical characterization and for biological detection.