Download Tumor necrosis factor (TNF)-α, also called TNFSF2 and Cachectin

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.