Identification of G-quadruplex anti-Interleukin-2 aptamer with high specificity through SELEX stringency

Aptamers are short single-stranded oligonucleotides capable of binding to various targets with high specificity and affinity. This study aimed to identify an aptamer against mouse interleukin-2 (mIL-2) as one of the most important cytokines in autoimmune diseases for diagnostic and therapeutic purposes. For this purpose, 14 SELEX rounds were performed on recombinant mIL-2 with high stringency. The dot blot and flow cytometry techniques were conducted to determine affinity, dissociation constant (K_d), specificity, and SELEX rounds screening. The stringency of rounds was considered based on aptamer/target incubation time, washing steps, and target proteins. Finally, the aptamer's structure was mapped and predicted by M-fold and QGRS Mapper web-based software. After 14 rounds, the flow cytometry analysis revealed that the 11^th round was a proper round. The high-affinity aptamers M20 and M15 were chosen for their ability to bind mIL-2. According to DNA folding software, M20 and M15 aptamers had G-quadruplex and stem-loop structures, respectively. The M20 aptamer affinity was greater than M15, and its predicted K_d was 91 nM. A simple SELEX protocol with round stringency was explained to identify DNA aptamers against protein targets. The reported G-quadruplex aptamer might have potential diagnostic or therapeutic application in IL-2-related disorders.