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Title: 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:
 [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:
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)
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. 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. Mon . "De novo DNA synthesis using polymerase-nucleotide conjugates". United States. doi: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 = {2018},
month = {6}
}

Journal Article:
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Cited by: 14 works
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Figures / Tables:

Figure 1 Figure 1: TdT–dNTP conjugates for reversible termination of primer elongation. (a) Scheme for two-step oligonucleotide extension using TdT–dNTP conjugates consisting of a TdT molecule site-specifically labeled with a dNTP via a cleavable linker. In the extension step, a DNA primer is exposed to an excess of TdT–dNTP conjugate. Upon incorporationmore » of the tethered nucleotide into the 3′ end of the primer, the conjugate becomes covalently attached and prevents further extensions by other TdT–dNTP molecules. In the deprotection step, the remaining TdT–dNTP conjugates are inactivated (or removed) and the linkage between the incorporated nucleotide and TdT is cleaved by addition of the cleavage reagent (e.g., DTT, 365 nm light, peptidase), thereby releasing the primer for subsequent extension. The cycle can be iterated to extend a primer by a defined sequence. (b) Chemical structures of two types of TdT–linker-dNTP conjugates used in this study, based on the amine-to-thiol crosslinkers PEG4-SPDP (upper, “TdT–PEG4-dTTP”) and BP-23354 (lower, “TdT–dTTP”) and the dTTP analogs 5-aminoallyl dUTP (aa-dUTP) and 5-propargylamino dUTP (pa-dUTP), respectively. Upon cleavage of the linker, the atoms indicated in red remain attached to the nucleobase and are referred to as a scar. The cleavable bond is indicated with a black dotted line.« less

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