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Title: De novo DNA synthesis using polymerase-nucleotide conjugates

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:
 [1] ;  [2] ;  [2] ;  [1] ;  [2] ;  [2] ;  [2] ;  [3] ;  [4] ;  [4] ; ORCiD logo [5] ; ORCiD logo [6]
  1. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Technische Univ. Darmstadt, Darmstadt (Germany)
  2. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States)
  3. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  4. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Sandia National Lab. (SNL-CA), Livermore, CA (United States)
  5. 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)
  6. 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:
Grant/Contract Number:
AC02-05CH11231
Type:
Accepted Manuscript
Journal Name:
Nature Biotechnology
Additional Journal Information:
Journal Volume: 36; Journal Issue: 7; Journal ID: ISSN 1087-0156
Publisher:
Springer Nature
Research Org:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES
OSTI Identifier:
1461176

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., 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. 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., and Keasling, Jay D.. 2018. "De novo DNA synthesis using polymerase-nucleotide conjugates". United States. doi:10.1038/nbt.4173.
@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 = {2018},
month = {6}
}

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