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

Abstract

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.

Modified Nucleic Acids: Expanding the Capabilities of Functional Oligonucleotides

“…While some literature does broaden this definition to include, for example, single-atom substitutions to any portion of the tripartite nucleotide [ 18 ], this subsection focuses specifically on sugar chemistries that are highly divergent from the traditional ribofuranose or deoxyribofuranose ring and thus bear minimal resemblance to their natural counterparts. For example, in lieu of the furanose ring, researchers have incorporated sugar groups such as threose in TNA [ 80 ] and even six membered ring structures like hexitol in HNA [ 12 ] illustrated in Figure 1 . Polymerase evolution experiments have provided the means to incorporate several of these XNAs such as cyclohexene in CeNA and TNA within in vitro evolutionary schemes [ 8 ].…”

In Vitro Selection of an ATP-Binding TNA Aptamer

“…Despite having a backbone repeat unit that is one atom shorter than the repeat unit found in natural DNA and RNA, TNA is capable of forming antiparallel Watson–Crick duplex structures with DNA, RNA, and itself [ 17 , 18 ]. Although TNA aptamers have been generated against several protein targets [ 19 , 20 , 21 ], very little is known about the ability for TNA to recognize small molecule targets with high affinity and specificity [ 22 ]. Here, we report the in vitro selection of an ATP-binding TNA aptamer that shows high specificity against other nucleotide triphosphates and strong resistance to nuclease degradation.…”

Selection of Aptamers Specific for DEHP Based on ssDNA Library Immobilized SELEX and Development of Electrochemical Impedance Spectroscopy Aptasensor

“…Aptamer 31 had the highest affinity with Kd = 2.26 ± 0.06 nM, which was lower than that for all reported aptamers specific for small molecules. In a previous report, the affinity constants were beyond 10 nM for most aptamers [31,32]. Generally, LSPR is a powerful and sensitive tool for elucidation of the interaction between DNA and small molecules [33].…”

“…First, 100 µg DEHP modified with carboxyl group (Figure 8) was fixed to the NH 2 chip through NH 2 and COOH interaction and then blocked with 1 M ethanolamine (pH 8.5). After the baseline was adjusted with phosphate buffer solution (PBS, 29.2 g NaCl, 0.69 g NaH 2 PO 4 , 0.71 g Na 2 HPO 4 , 0.1 g MgCl 2 ·6H 2 O; 500 mL, pH 7.0), aptamers (31,123,203,281) diluted to different concentrations (2.5, 5, 10, and 20 or 5, 10, 20, and 40 nM) were allowed to bind with DEHP for 240s. The flowrate was 20 µL/min, and the chip was regenerated with 5-10 mM NaOH.…”