In vitro selection of an XNA aptamer capable of small-molecule recognition

Despite advances in XNA evolution, the binding capabilities of artificial genetic polymers are currently limited to protein targets. Here, we describe the expansion of in vitro evolution techniques to enable selection of threose nucleic acid (TNA) aptamers to ochratoxin A (OTA). This research establishes the first example of an XNA aptamer of any kind to be evolved having affinity to a small-molecule target. Selection experiments against OTA yielded aptamers having affinities in the mid nanomolar range; with the best binders possessing KD values comparable to or better than those of the best previously reported DNA aptamer to OTA. Importantly, the TNA can be incubated in 50% human blood serum for seven days and retain binding to OTA with only a minor change in affinity, while the DNA aptamer is completely degraded and loses all capacity to bind the target. This not only establishes the remarkable biostability of the TNA aptamer, but also its high level of selectivity, as it is capable of binding OTA in a large background of competing biomolecules. Together, this research demonstrates that refining methods for in vitro evolution of XNA can enable the selection of aptamers to a broad range of increasingly challenging target molecules.