Post-SELEX modifications with locked nucleic acids (LNA) of a SEA-specific DNA aptamer assisted by in silico modelling†

Post-SELEX modifications assisted by in silico modelling are powerful tools to improve the performance of aptamers, by providing a rational approach for the selection of modified-versions of aptamers. In this study, a complete in silico analysis of the three-dimensional structure of a previously selected DNA aptamer (Apt5) against staphylococcal enterotoxin A (SEA) was performed. Locked nucleic acid (LNA) modifications were introduced in key locations and their effect on the aptamer structure and docking were evaluated. Promising LNA aptamers were then synthetized and their dissociation constants (K_D), as well as stability, were evaluated. From the in silico analysis, it was possible to identify three promising LNA variations that did not affect drastically the three-dimensional structure and the molecular docking with the toxin. The K_D of the LNA aptamers were higher than the DNA aptamer (Apt5: K_D = 13 ± 2 nM, LNA13: K_D = 157 ± 39 nM, LNA14: K_D = 74 ± 24 nM, LNA15: K_D = 143 ± 28 nM), but remained in the low nanomolar range. Even so, the K_D of LNA14 was not significantly different (P < 0.05) compared to the value of the original aptamer and the introduction of LNA increased its thermal stability, increasing the range of functionality of the original aptamer. However, the introduced modifications were not enough to increase the biological stability of the aptamer, remaining susceptible to a complete degradation by endonucleases and exonucleases in 5 minutes. Altough partial modifications with LNA may not be able to overcome all the limitations of DNA aptamers, post-SELEX modifications assisted by in silico modelling have shown promising results in predicting functional modified aptamers, avoiding a time-consuming and expensive trial and error approach.