Identification and characterization of a functional DcR3 aptamer for diagnostic and therapeutic applications
M-F. Hsueh, C-L. Lee, J-H. Huang, W-C. Kao, K. Peck
Academia Sinica, TW
Keywords: DcR3, aptamer, protein detection
Abstract:DcR3, a soluble decoy receptor that belongs to the tumor necrosis factor receptor (TNFR) superfamily, binds to Fas ligand and inhibits Fas ligand-induced apoptosis. Aptamers that recognize DcR3 were identified by a Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process. These aptamers were optimized by a microarray-based method to achieve high affinity and specificity. The dissociation constants (Kd) of these optimized aptamers were less than 3 nM, showing that the SELEX process and the post-SELEX optimization developed by us worked well. One of the DcR3 aptamers was characterized to have antagonistic activity against target protein by using Fas-sensitive Jurkat cell. Our experimental results showed that apoptosis of Jurkat cells was induced by activated Fas ligand but was significantly inhibited with the presence of DcR3. However, the apoptosis inhibition was abrogated by the addition of the DcR3 antagonistic aptamer. In addition, we have developed an aptamer based assay platform dubbed ELONA for in vitro diagnosis. The detection limit of the ELONA for DcR3 was as low as 2 pg/ml. The dynamic range of the assay has a span of three orders of magnitude with the coefficient of determination r2 = 0.997.