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<p>Discovery of functional nucleic acids from randomized libraries typically relies on multiple, time-consuming iterative rounds of in vitro selection with low success rate. Here, we present a single-round selection strategy for rapid screening of multiple over-represented nucleobase-modified DNA libraries and various selection conditions, capable of identifying high-affinity modified aptamers. Double partition followed by amplification of eluted sequences, NGS analysis and clustering provides fast identification of aptamer candidates. Screening of modified DNA libraries containing modified adenine and uracil nucleotides bearing hydrophobic aromatic phenyl and indole moieties results in development of an aptamer binding human insulin receptor with sub-nanomolar affinity and exquisite specificity. Cryo-EM structure reveals the importance of each aromatic modification, either in stabilizing the secondary structure or facilitating interactions with the protein surface. This approach addresses the main drawbacks of aptamer selection and has potential for high-throughput screening and accelerating the development of next-generation aptamers for diagnostics or therapeutics.</p>

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Expedient single-round selection of hyper-modified aptamer targeting insulin receptor from over-represented dually nucleobase-modified DNA libraries

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年5月27日

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  • Pablo Alberto Franco-Urquijo,
  • Marek Ondruš,
  • Jaroslav Kurfürst,
  • Jana Škerlová,
  • Irena Selicharová,
  • Lucie Mužíková Čechová,
  • Hana Šváchová,
  • Alena Semerádtová,
  • Anatolij Filimoněnko,
  • Adéla Fejfarová,
  • Jiří Homola,
  • Tomáš Kouba,
  • Michal Hocek

摘要

Discovery of functional nucleic acids from randomized libraries typically relies on multiple, time-consuming iterative rounds of in vitro selection with low success rate. Here, we present a single-round selection strategy for rapid screening of multiple over-represented nucleobase-modified DNA libraries and various selection conditions, capable of identifying high-affinity modified aptamers. Double partition followed by amplification of eluted sequences, NGS analysis and clustering provides fast identification of aptamer candidates. Screening of modified DNA libraries containing modified adenine and uracil nucleotides bearing hydrophobic aromatic phenyl and indole moieties results in development of an aptamer binding human insulin receptor with sub-nanomolar affinity and exquisite specificity. Cryo-EM structure reveals the importance of each aromatic modification, either in stabilizing the secondary structure or facilitating interactions with the protein surface. This approach addresses the main drawbacks of aptamer selection and has potential for high-throughput screening and accelerating the development of next-generation aptamers for diagnostics or therapeutics.