Cited by 316DNA and Nucleic Acid ChemistryAdvanced biosensing and bioanalysis techniquesEnzyme Catalysis and Immobilization

De novo DNA synthesis using polymerase-nucleotide conjugates.

Open access
Palluk, Sebastian, Arlow, Daniel H, de Rond, Tristan, Barthel, Sebastian, Kang, Justine S, Bector, Rathin, Baghdassarian, Hratch M, Truong, Alisa N +4 more·2018·Nature Biotechnology

Abstract

Oligonucleotides are almost exclusively synthesized using the nucleoside phosphoramidite method, even though it is limited to the direct synthesis of ∼200 mers and produces hazardous waste. Here, we describe an oligonucleotide synthesis strategy that uses the template-independent polymerase terminal deoxynucleotidyl transferase (TdT). Each TdT molecule is conjugated to a single deoxyribonucleoside triphosphate (dNTP) molecule that it can incorporate into a primer. After incorporation of the tethered dNTP, the 3' end of the primer remains covalently bound to TdT and is inaccessible to other TdT-dNTP molecules. Cleaving the linkage between TdT and the incorporated nucleotide releases the primer and allows subsequent extension. We demonstrate that TdT-dNTP conjugates can quantitatively extend a primer by a single nucleotide in 10-20 s, and that the scheme can be iterated to write a defined sequence. This approach may form the basis of an enzymatic oligonucleotide synthesizer.

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Crossref · 10.1038/nbt.4173

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Authors
Palluk, S., Arlow, D. H., de Rond, T., Barthel, S., Kang, J. S., Bector, R., Baghdassarian, H. M., Truong, A. N., Kim, P. W., Singh, A. K., Hillson, N. J., & Keasling, J. D.
Affiliations
  • Lawrence Berkeley National Laboratory (US)
  • Sandia National Laboratories California (US)
Corresponding author
  • Arlow, Daniel H
  • Keasling, Jay D
Venue
Nature Biotechnology
Published
2018-06-18
Volume
36
Issue
7
Pages
645-650
ISSN
  • 0733-222X (ISSN-L)
  • 1087-0156
  • 1546-1696
OpenAlex
W2808129035
Cited by
316
References
41
Citations by year
2026: 39 · 2025: 49 · 2024: 61 · 2023: 43 · 2022: 27 · 2021: 21 · 2020: 33 · 2019: 31 · 2018: 11 · 2012: 1
FWCI
8.54
Type
journal article
Language
en
Locations
  • Nature Biotechnology (publishedVersion)
  • PubMed (publishedVersion)
  • Technischen Universität Darmstadt (acceptedVersion, cc-by-nc-sa)
  • OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion)
  • eScholarship (California Digital Library) (submittedVersion)
  • TUbilio (Technical University of Darmstadt) (submittedVersion, cc-by-nc-sa)
  • Technischen Universität Darmstadt
Funders
Novo Nordisk Fonden (NNF10CC1016517)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Advanced biosensing and bioanalysis techniques
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
Keywords
terminal deoxynucleotidyl transferase, enzymatic oligonucleotide synthesis, primer extension, deoxyribonucleoside triphosphates, enzymatic DNA synthesis
MeSH terms
DNA Nucleotidylexotransferase, DNA-Directed DNA Polymerase, DNA Replication, Nucleosides, Oligonucleotides, Organophosphorus Compounds

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