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.

Synthesis and nonenzymatic template-directed polymerization of 2'-amino-2'-deoxythreose nucleotides.

Threose nucleic acid (TNA) is a potential alternative genetic material that may have played a role in the early evolution of life. We have developed a novel synthesis of 2'-amino modified TNA nucleosides (2'-NH2-TNA) based on a cycloaddition reaction between a glycal and an azodicarboxylate, followed by direct nucleosidation of the cycloadduct. Using this route, we synthesized the thymine and guanine 2'-NH2-TNA nucleosides in seven steps with 24% and 12% overall yield, respectively. We then phosphorylated the guanine nucleoside on the 3'-hydroxyl, activated the phosphate as the 2-methylimidazolide, and tested the ability of the activated nucleotide to copy C4 RNA, DNA, and TNA templates by nonenzymatic primer extension. We measured pseudo-first-order rate constants for the first nucleotide addition step of 1.5, 0.97, and 0.57 h(-1) on RNA, DNA, and TNA templates, respectively, at pH 7.5 and 4 °C with 150 mM NaCl, 100 mM N-(hydroxylethyl)imidazole catalyst, and 5 mM activated nucleotide. The activated nucleotide hydrolyzed with a rate constant of 0.39 h(-1), causing the polymerization reaction to stall before complete template copying could be achieved. These extension rates are more than 1 order of magnitude slower than those for amino-sugar ribonucleotides under the same conditions, and copying of the TNA template, which best represented a true self-copying reaction, was the slowest of all. The poor kinetics of 2'-NH2-TNA template copying could give insight into why TNA was ultimately not used as a genetic material by biological systems.

Darwinian evolution of an alternative genetic system provides support for TNA as an RNA progenitor.

The pre-RNA world hypothesis postulates that RNA was preceded in the evolution of life by a simpler genetic material, but it is not known if such systems can fold into structures capable of eliciting a desired function. Presumably, whatever chemistry gave rise to RNA would have produced other RNA analogues, some of which may have preceded or competed directly with RNA. Threose nucleic acid (TNA), a potentially natural derivative of RNA, has received considerable interest as a possible RNA progenitor due to its chemical simplicity and ability to exchange genetic information with itself and RNA. Here, we have applied Darwinian evolution methods to evolve, in vitro, a TNA receptor that binds to an arbitrary target with high affinity and specificity. This demonstration shows that TNA has the ability to fold into tertiary structures with sophisticated chemical functions, which provides evidence that TNA could have served as an ancestral genetic system during an early stage of life.