Synthesis and Evolution of a Threose Nucleic Acid Aptamer Bearing 7-Deaza-7-Substituted Guanosine Residues

Abstract

In vitro selection experiments carried out on artificial genetic polymers require robust and faithful methods for copying genetic information back and forth between DNA and xeno-nucleic acids (XNA). Previously, we have shown that Kod-RI, an engineered polymerase developed to transcribe DNA templates into threose nucleic acid (TNA), can function with high fidelity in the absence of manganese ions. However, the transcriptional efficiency of this enzyme diminishes greatly when individual templates are replaced with libraries of DNA sequences, indicating that manganese ions are still required for in vitro selection. Unfortunately, the presence of manganese ions in the transcription mixture leads to the misincorporation of tGTP nucleotides opposite dG residues in the templating strand, which are detected as G-to-C transversions when the TNA is reverse transcribed back into DNA. Here we report the synthesis and fidelity of TNA replication using 7-deaza-7-modified guanosine base analogues in the DNA template and incoming TNA nucleoside triphosphate. Our findings reveal that tGTP misincorporation occurs via a Hoogsteen base pair in which the incoming tGTP residue adopts a syn conformation with respect to the sugar. Substitution of tGTP for 7-deaza-7-phenyl tGTP enabled the synthesis of TNA polymers with >99% overall fidelity. A TNA library containing the 7-deaza-7-phenyl guanine analogue was used to evolve a biologically stable TNA aptamer that binds to HIV reverse transcriptase with low nanomolar affinity.

In Vitro Selection of an ATP-Binding TNA Aptamer

“…Since the amount of oligonucleotide required for this technique is not easily achieved by enzymatic synthesis, we decided to generate the truncated aptamer by solid-phase synthesis using TNA phosphoramidite chemistry [ 26 ]. The TNA amidites for the natural bases were prepared using previously established methodology [ 27 ], while the 7-phenyl-7-deaza-tG phosphoramidite monomer was prepared ( Scheme 1 ) using a slightly modified version of the strategy that was established previously for the triphosphate version of the 7-phenyl-7-deaza-tG monomer [ 20 ]. The synthetic oligonucleotide obtained for T10-7.t5 yields a binding isotherm with a K D value of 22 ± 5 μM ( Figure 3 a), which is comparable to DNA aptamers that were evolved previously to bind ATP [ 9 ].…”

“…Kod-RI TNA polymerase and Bst-LF DNA polymerase were expressed and purified as described previously [ 30 , 31 ]. TNA triphosphates bearing natural bases and 7-phenyl-7-deaza-guanine were synthesized as previously described [ 20 , 27 , 32 ]. DNA oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA, USA), purified by denaturing polyacrylamide gel electrophoresis (PAGE), electroeluted, buffer exchanged and concentrated using Millipore YM-10 or YM-30 Centricon centrifugal devices, and quantified by UV absorbance.…”

“…TNA phosphoramidites with standard bases were synthesized as previously described [ 26 ]. The 7-deaza-7-phenyl-tG phosphoramidite was synthesized based on a previously established protocol for the synthesis of 7-deaza-7-phenyl tGTP monomer [ 20 ]. Standard β-cyanoethyl phosphoramidite chemistry and an Applied Biosystems 3400 DNA Synthesizer were used to synthesize TNA oligonucleotides on Universal Support II CPG columns (1 μM scale, Glen Research, Sterling, VA, USA).…”

“…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.…”

Click-PD: A Quantitative Method for Base-Modified Aptamer Discovery

“…3,6 More recently, other groups have modified the backbones of nucleic acids to generate 'xenobiotic' nucleic acid (XNA) aptamers with novel chemical functionalities 7 , including exceptional stability in complex biological milieus. 8,9 The process of generating base-modified aptamers typically entails the use of engineered polymerases that can faithfully incorporate and amplify both natural and chemically-modified nucleotides while maintaining minimal error rates. 10 Unfortunately, in addition to the considerable time and resources associated with engineering such polymerases, the resulting enzymes may not exhibit sufficient processivity or fidelity for efficient aptamer selection, 11,12 and each new chemical modification may require a new campaign of polymerase engineering.…”