Optimization And Verification Of A Specific STX Aptamer

Saxitoxin (STX), a member of the family of paralytic shellfish poisoning toxins, which is synthesized by marine algae, can be enriched in crustaceans and mollusks through the food chain, leading to human and marine animals paralytic shellfish poisoning after ingestion. In order to protect human from poisoning, it is very important to monitor the amount of STX. However, the most critical part in the monitoring programmes for STX is its molecular recognition element. Aptamers, which are short oligonucleotides (DNA or RNA), can specifically bind with a variety of targets, such as drugs, proteins, other inorganic or organic molecules, with high affinity. They have a variety of advantages compared to antibodies, including their stability, low immunogenicity, easy chemical synthesis, etc., and have been used as molecular recognition element in the target detection, disease diagnosis, disease treatment and other aspects for application. To develop an alternative molecular recognition element for STX, a DNA aptamer (APTSTX1) was previously discovered via an iterative process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX) by Handy, et al. However, characterizations of its kinetic parameter, and function, and post-SELEX optimization, which are necessary to improve its characteristics for further application, have not been performed yet.It sometimes fails to identify aptamers that bind with their target with high affinity, because of amplification bias of PCR and reduced library diversity caused by experimental manipulation. Different groups have engineered mutated aptamers that bind to targets with improved affinity compared to the wild type. We presume that if an aptamer can form a G-quadruplex after slight mutation, its affinity could be improved based on its strengthened structural stability. Since sets of guanine bases are necessary for G-quadruplex structures, we first designed a number of mutant sequences, with mutation sites at both sides of guanine bases, which were replaced by guanine nucleotide. After the prediction by QGRS Mapper, we synthesized the G-quadruplex forming sequences and evaluated their STX-binding ability by Bio-Layer Interferometry. Among all the mutated sequences, some performed improved affinity, with M-30 having the highest affinity,30 folds that of APTSTX1. Then, we conducted truncation analysis on M-30, with the ends and the stem-loop structures removed one by one. Kd values of those truncated sequences were also calculated by Bio-Layer Interferometry. Through the research on the relationship between structure and affinity of truncated aptamers, the STX-binding core structure was identified to be the two stem-loop structures at the 5’end. After removing the excess nucleotides, we obtain M-30f, with a Kd value of 133nM by Bio-Layer Interferometry. Finally, through a variety of methods (ELISA, cell bioassay and MBA), we compared the affinity of the two aptamers before and after optimization (APTSTX1 and M-30f) and evaluated their inhibitory activity against STX. Both aptamers’STX-binding ability was demonstrated in all three methods.In the ELISA and cell bioassays, statistically significant differences could be observed between their interactions with STX. Moreover, the suppressive activity of STX on sodium channels could be inhibited by both aptamers, with M-30f performing better according to the cell bioassay.As indicated, M-30f performs better than its parent sequence and has good prospects for practical application as a molecular recognition element. Moreover, the study demonstrates that as an alternative strategy for post-SELEX optimization, it is feasible to generate improved aptamers by rational site-directed mutagenesis and truncation.