The Protein-Templated Synthesis of Enzyme-Generated Aptamers

Julio Vacacela, Anna-Lisa Schaap-Johansen, Patricia Manikova, Paolo Marcatili, Marta Prado, Yi Sun, Jon Ashley*

*Corresponding author for this work

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Inspired by the chemical synthesis of molecularly imprinted polymers, we demonstrated for the first time, the protein-target mediated synthesis of enzyme-generated aptamers (EGAs). We prepared pre-polymerisation mixtures containing different ratios of nucleotides, an initiator sequence and protein template and incubated each mixture with terminal deoxynucleotidyl transferase (TdT). Upon purification and rebinding of the EGAs against the target, we observed an enhancement in binding of templated-EGAs against the target compared to a non-templated control. These results demonstrate the presence of two primary mechanisms for the formation of EGAs, namely, the binding of random sequences to the target as observed in systematic evolution of ligands by exponential enrichment (SELEX) and the dynamic competition between TdT enzyme and the target protein for binding of EGAs during synthesis. The latter mechanism serves to increase the stringency of EGA-based screening and represents a new way to develop aptamers that relies on rational design.
Original languageEnglish
Article number e20220106
Number of pages5
Publication statusPublished - 2022


  • Terminal deoxynucleotidyl transferase
  • Enzyme
  • Protein
  • Aptamer
  • Enzyme-generated aptamers


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