Affinity Maturation of a Fentanyl Aptamer by Motif-SELEX

Oligonucleotide-based aptamers are increasingly being used as recognition elements in biosensors to detect analytes relevant to diverse applications including medical diagnostics and therapeutic drug monitoring. Nevertheless, the utility of aptamers is often hindered by their insufficient target-binding affinity. We recently developed an affinity maturation approach termed Motif-SELEX that can improve aptamer binding affinity by up to an order of magnitude. This approach entails performing in vitro selection with a library containing conserved sequence motifs of an existing aptamer flanked by variable-length random domains that serve to enhance interactions with the target. Here, we demonstrate the application of Motif-SELEX to a DNA aptamer that binds to fentanyl with highalbeit inadequateaffinity to enable the detection of fentanyl at clinically relevant concentrations in biofluids. The matured aptamers discovered through Motif-SELEX are somewhat more complex than previous-generation aptamers, with additional nucleobases in their binding domain, and display 3-fold improved affinity toward fentanyl. We then use these aptamers to develop molecular beacon sensors that can detect fentanyl at concentrations as low as 6 nM in diluted filtered serum. Our findings indicate that Motif-SELEX offers an effective means to improve the affinity of underperforming aptamers toward levels suitable for practical use.