Generation of Species Cross-reactive Aptamers Using “Toggle” SELEX

TLDR

Species cross‑reactivity enables preclinical testing of therapeutic molecules in animal models. The study aims to develop a toggle SELEX approach that yields aptamers binding both human and porcine thrombin. The method alternates selection rounds between human and porcine thrombin to enrich cross‑reactive RNA ligands. The toggle SELEX generated high‑affinity cross‑reactive aptamers, including Toggle‑25, which inhibited thrombin‑mediated clot formation and platelet activation, and the strategy is generalizable to other proteins and therapeutic applications.

Abstract

Species cross-reactivity facilitates the preclinical evaluation of potentially therapeutic molecules in animal models. Here we describe an in vitro selection strategy in which RNA ligands (aptamers) that bind both human and porcine thrombin were selected by "toggling" the protein target between human and porcine thrombin during alternating rounds of selection. The "toggle" selection process yielded a family of aptamers, all of which bound both human and porcine thrombin with high affinity. Toggle-25, a characteristic member, inhibited two of thrombin's most important functions: plasma clot formation and platelet activation. If appropriate targets are available, the toggle strategy is a simple measure that promotes cross-reactivity and may be generalizable to related proteins of the same species as well as to other combinatorial library screening strategies. This strategy should facilitate the isolation of ligands with needed properties for gene therapy and other therapeutic and diagnostic applications.