Highly Parallelized Screening of Functionally Enhanced XNA Aptamers in Uniform Hydrogel Particles

Xeno-nucleic acid (XNA) aptamers based on evolvable non-natural genetic polymers hold enormous potential as future diagnostic and therapeutic agents. However, time-consuming and costly procedures requiring the purification of individual XNA sequences produced by large-scale polymerase-mediated primer extension reactions pose a major bottleneck to the discovery of highly active XNA motifs for biomedical applications. Here, we describe a straightforward approach for rapidly surveying the binding properties of XNA aptamers identified by in vitro selection. Our strategy involves preparing XNA aptamer particles in which many copies of the same aptamer sequence are distributed throughout the gel matrix of a polyacrylamide-encapsulated magnetic particle. Aptamer particles are then screened by flow cytometry to assess target binding affinity and deduce structure-activity relationships. This generalizable and highly parallel assay dramatically accelerates the pace of secondary screening by allowing a single researcher to evaluate 48-96 sequences per day.