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De novo DNA synthesis using polymerase-nucleotide conjugates.
Open accessAbstract
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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Metadata
- 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
- 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
- FWCI
- 8.54
- Type
- journal article
- Language
- en
- Open access
- Licence
- 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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