In Vitro Selection of an ATP-Binding TNA Aptamer

Abstract

Recent advances in polymerase engineering have made it possible to isolate aptamers from libraries of synthetic genetic polymers (XNAs) with backbone structures that are distinct from those found in nature. However, nearly all of the XNA aptamers produced thus far have been generated against protein targets, raising significant questions about the ability of XNA aptamers to recognize small molecule targets. Here, we report the evolution of an ATP-binding aptamer composed entirely of α-L-threose nucleic acid (TNA). A chemically synthesized version of the best aptamer sequence shows high affinity to ATP and strong specificity against other naturally occurring ribonucleotide triphosphates. Unlike its DNA and RNA counterparts that are susceptible to nuclease digestion, the ATP-binding TNA aptamer exhibits high biological stability against hydrolytic enzymes that rapidly degrade DNA and RNA. Based on these findings, we suggest that TNA aptamers could find widespread use as molecular recognition elements in diagnostic and therapeutic applications that require high biological stability.

Keywords: TNA; aptamer; biological stability.

Figure 1

Selection of threose nucleic acid (TNA) aptamers. (a) Chemical structures of RNA and TNA. (b) tNTP substrates used to enzymatically synthesize TNA molecules for in vitro selection. (c) Cartoon image of the in vitro selection strategy used to isolate ATP-binding TNA aptamers.

Figure 2

ATP binding activity of in vitro selected TNA aptamers. TNA aptamers tagged with an IR680 dye were evaluated for their ability bind to and elute from ATP-derivatized agarose beads. (a) Activity screen of eight unique ATP binding aptamers isolated after 10 rounds of in vitro selection and amplification. (b) Truncation analysis of aptamer 10-7 using the technique of 5′ and 3′ end-mapping deletion analysis. (c) Binding controls evaluating the contribution of the 7-phenyl-guanine base to the binding activity of the full-length (10-7) and truncated (t5) versions of the TNA aptamer. Other controls included binding assays to underivatized agarose beads as well as the DNA and TNA primer only sequences to ATP derivatized agarose.

Scheme 1

Chemical Synthesis of 7-deaza-7-phenyl guanosine TNA phosphoramidite monomer used for solid-phase synthesis.

Figure 3

Solution binding affinity (Kd) of the ATP-binding TNA aptamer to free ATP and other closely related nucleoside triphosphates. All binding assays were performed using the minimal sequence (10-7.t5) TNA aptamer prepared by solid-phase synthesis. Binding measurements were performed in binding buffer containing 5 mM MgCl₂, 300 mM NaCl, and 20 mM Tris-HCl (pH 7.6) at 24 °C. (a) Aptamer sequence. (b) Binding isotherm obtained for ATP. (c) Summary of Kd values obtained for other nucleoside triphosphates and ATP analogs.

Figure 4

Biological stability assays. Time-dependent assays were performed using snake venom phoshpodiesterase (SVPDE) and human liver microsome (HLM) to evaluate the biological stability of ATP-binding TNA and DNA aptamers. M is a marker for the 0-time point with no enzyme.