Triple SELEX approach for the selection of a highly specific RNA aptamer binding homoeriodictyol.

The application of synthetic riboswitches or aptamer-based biosensors for the monitoring of engineered metabolic pathways greatly depends on a high degree of target molecule specificity. Since metabolic pathways include close derivatives that often differ only in single moieties, the binding specificity of aptamers utilized for these systems has to be high. In the present study, we selected an RNA aptamer that is highly specific in its binding to homoeriodictyol while discriminating its close derivatives eriodictyol and naringenin. This high degree in specificity was achieved through three consecutive SELEX approaches while the selection parameters were adjusted and refined from one to the next. The adjustments along the process, with the selection outcome and next-generation sequencing analysis of the selection rounds, led to valuable insights into the stringency necessary to facilitate target specificity in aptamers obtained from SELEX. From the third selection, we obtained a highly binding specific aptamer and examined its structure and binding properties. Overall, our results connect the importance of selection stringency with SELEX outcome and aptamer specificity while providing a highly selective, homoeriodictyol-binding RNA aptamer.