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PreprintCC BYChemRxiv

Evaluation of alternative polymerases for biocatalytic synthesis of modified oligonucleotides

  • Fabienne Levi-Acobas1
  • Germain Niogret1,2
  • Sophie Battédou1
  • Gilles Gasser2
  • Markel Martinez Carranza3,4
  • Marc Delarue3,4
  • Ludovic Sauguet3,4
  • Marcel Hollenstein1
4 affiliations
  1. 1Department of Structural Biology and Chemistry, Laboratory for Bioorganic Chemistry of Nucleic Acids, 28, rue du Docteur Roux, Paris Cedex, France, 75724
  2. 2Chimie ParisTech, PSL University, CNRS, Institute of Chemistry for Life and Health Sciences, Laboratory for Inorganic Chemical Biology
  3. 3Institut Pasteur
  4. 4Unit of Architecture and Dynamics of Biological Macromolecules
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Abstract

Enzymatic synthesis of modified nucleic acids is gaining a lot of traction. Most approaches involve the combination of modified nucleoside triphosphates and engineered polymerases capable of accepting such altered substrates. Such enzymes are obtained through engineering campaigns based on directed evolution, positional saturation mutagenesis, and rational design approaches potentially guided by in silico methods. However, most polymerase evolution/design approaches rely on a restricted diversity of enzyme chassis. Here, we have performed a biochemical characterization of two polymerases, the human DNA HsPolθ and the heterodimeric archaeal DNA primase PriSL from Pyrococcus abyssi, capable of synthesizing oligonucleotides under both template-dependent and -independent reaction conditions. We have assessed their capacity at accepting nucleotides with modifications at any position of the scaffold as well as reversible terminators. We have also evaluated the possibility of using high salt conditions to force polymerases to incorporate single nucleotides. Overall, our study reveals that both polymerases are tolerant to modifications and could be amenable to engineering campaigns to identify variants capable of producing hypermodified XNAs for de novo nucleic acid synthesis approaches.

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Cite

BibTeXRIS
BibTeX
@misc{leviacobas2026evaluation,
  title = {{Evaluation of alternative polymerases for biocatalytic synthesis of modified oligonucleotides}},
  author = {Fabienne Levi-Acobas and Germain Niogret and Sophie Battédou and Gilles Gasser and Markel Martinez Carranza and Marc Delarue and Ludovic Sauguet and Marcel Hollenstein},
  howpublished = {ChemRxiv},
  year = {2026},
  month = oct,
  doi = {10.26434/chemrxiv.15009757/v1},
  url = {https://chemrxiv.org/doi/full/10.26434/chemrxiv.15009757/v1},
  note = {CC BY},
}
RIS
TY  - UNPB
TI  - Evaluation of alternative polymerases for biocatalytic synthesis of modified oligonucleotides
AU  - Fabienne Levi-Acobas
AU  - Germain Niogret
AU  - Sophie Battédou
AU  - Gilles Gasser
AU  - Markel Martinez Carranza
AU  - Marc Delarue
AU  - Ludovic Sauguet
AU  - Marcel Hollenstein
PY  - 2026
DA  - 2026/10/01
PB  - ChemRxiv
DO  - 10.26434/chemrxiv.15009757/v1
UR  - https://chemrxiv.org/doi/full/10.26434/chemrxiv.15009757/v1
AB  - Enzymatic synthesis of modified nucleic acids is gaining a lot of traction. Most approaches involve the combination of modified nucleoside triphosphates and engineered polymerases capable of accepting such altered substrates. Such enzymes are obtained through engineering campaigns based on directed evolution, positional saturation mutagenesis, and rational design approaches potentially guided by in silico methods. However, most polymerase evolution/design approaches rely on a restricted diversity of enzyme chassis. Here, we have performed a biochemical characterization of two polymerases, the human DNA HsPolθ and the heterodimeric archaeal DNA primase PriSL from Pyrococcus abyssi, capable of synthesizing oligonucleotides under both template-dependent and -independent reaction conditions. We have assessed their capacity at accepting nucleotides with modifications at any position of the scaffold as well as reversible terminators. We have also evaluated the possibility of using high salt conditions to force polymerases to incorporate single nucleotides. Overall, our study reveals that both polymerases are tolerant to modifications and could be amenable to engineering campaigns to identify variants capable of producing hypermodified XNAs for de novo nucleic acid synthesis approaches.
ER  -