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

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De novo DNA synthesis using polymerase-nucleotide conjugates.

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Palluk, Sebastian, Arlow, Daniel H, de Rond, Tristan, Barthel, Sebastian, Kang, Justine S, Bector, Rathin, Baghdassarian, Hratch M, Truong, Alisa N, Kim, Peter W, Singh, Anup K, Hillson, Nathan J, Keasling, Jay D·2018·Nature Biotechnology

DOI 10.1038/nbt.4173

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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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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
ORCID: Arlow, Daniel H
Barthel, Sebastian
Baghdassarian, Hratch M
Kim, Peter W
Singh, Anup K
Hillson, Nathan J
Keasling, Jay D
Venue: Nature Biotechnology
Publisher: Springer Science and Business Media LLC
Published: 2018-06-18
Volume: 36
Issue: 7
Pages: 645-650
ISSN: 0733-222X (ISSN-L)
1087-0156
1546-1696
DOI: 10.1038/nbt.4173
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
FWCI: 8.54
Type: journal article
Language: en
Open access: green
tuprints.ulb.tu-darmstadt.de
Licence: cc-by-nc-sa
www.springernature.com/gp/researchers/text-and-data-mining (tdm)
www.springernature.com/gp/researchers/text-and-data-mining (vor)
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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De novo DNA synthesis using polymerase-nucleotide conjugates.

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