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

Abstract

Xeno-nucleic acid (XNA) aptamers based on evolvable nonnatural 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 largescale 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.

Functionally Enhanced XNA Aptamers Discovered by Parallelized Library Screening

“…To illuminate the relationship between library chemotype and function, 96 representative members from each chemotype family were evaluated for binding to S1-RBD using a highly parallel hydrogel aptamer particle strategy . In contrast to traditional particle display, whereby DNA aptamers are attached directly to the surface of a magnetic particle using an emulsion PCR and template stripping protocol, hydrogel aptamer particles are prepared by extension of a DNA primer that has been cross-linked into the gel matrix of a polyacrylamide hydrogel shell encapsulating a magnetic particle . The TNA aptamer is generated by primer extension using a DNA template encoding the desired TNA sequence.…”

“…The resulting monoclonal TNA aptamer hydrogel particles are evaluated for binding to a biotin-modified version of S1-RBD in 96-well format using an ELISA-type assay that is compatible with flow cytometry (Figure a). In this assay, aptamers with affinity to S1-RBD fluoresce at levels indicative of their binding affinity when incubated with phycoerythrin (PE)-labeled streptavidin (SA) . Consequently, aptamers exhibiting high mean fluorescence values relative to aptamer-deficient hydrogel particles (DNA primer only), viewed here as a background control, are predicted to function with a higher target binding affinity due to their ability to remain bound to the target protein following iterative wash steps.…”