De novo DNA synthesis using polymerase-nucleotide conjugates
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. Furthermore, this approach may form the basis of an enzymatic oligonucleotide synthesizer.
- Authors:
-
- Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Technische Univ. Darmstadt, Darmstadt (Germany)
- Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States)
- Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
- Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Sandia National Lab. (SNL-CA), Livermore, CA (United States)
- Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States)
- Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States); Technical Univ. of Denmark, Horsholm (Denmark)
- Publication Date:
- Research Org.:
- Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER)
- OSTI Identifier:
- 1461176
- Grant/Contract Number:
- AC02-05CH11231
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Nature Biotechnology
- Additional Journal Information:
- Journal Volume: 36; Journal Issue: 7; Journal ID: ISSN 1087-0156
- Publisher:
- Springer Nature
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES
Citation Formats
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., and Keasling, Jay D. De novo DNA synthesis using polymerase-nucleotide conjugates. United States: N. p., 2018.
Web. doi:10.1038/nbt.4173.
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. De novo DNA synthesis using polymerase-nucleotide conjugates. United States. https://doi.org/10.1038/nbt.4173
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., and Keasling, Jay D. Mon .
"De novo DNA synthesis using polymerase-nucleotide conjugates". United States. https://doi.org/10.1038/nbt.4173. https://www.osti.gov/servlets/purl/1461176.
@article{osti_1461176,
title = {De novo DNA synthesis using polymerase-nucleotide conjugates},
author = {Palluk, Sebastian and Arlow, Daniel H. and de Rond, Tristan and Barthel, Sebastian and Kang, Justine S. and Bector, Rathin and Baghdassarian, Hratch M. and Truong, Alisa N. and Kim, Peter W. and Singh, Anup K. and Hillson, Nathan J. and Keasling, Jay D.},
abstractNote = {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. Furthermore, this approach may form the basis of an enzymatic oligonucleotide synthesizer.},
doi = {10.1038/nbt.4173},
journal = {Nature Biotechnology},
number = 7,
volume = 36,
place = {United States},
year = {Mon Jun 18 00:00:00 EDT 2018},
month = {Mon Jun 18 00:00:00 EDT 2018}
}
Web of Science
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