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Title: Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3′ Terminal Structures for Enzymatic De Novo DNA Synthesis

Abstract

Enzymatic oligonucleotide synthesis methods based on the template-independent polymerase terminal deoxynucleotidyl transferase (TdT) promise to enable the de novo synthesis of long oligonucleotides under mild, aqueous conditions. Intermediates with a 3' terminal structure (hairpins) will inevitably arise during synthesis, but TdT has poor activity on these structured substrates, limiting its usefulness for oligonucleotide synthesis. Here, we described two parallel efforts to improve the activity of TdT on hairpins: (1) optimization of the concentrations of the divalent cation cofactors and (2) engineering TdT for enhanced thermostability, enabling reactions at elevated temperatures. By combining both of these improvements, we obtained a ~10-fold increase in the elongation rate of a guanine-cytosine hairpin.

Authors:
ORCiD logo [1];  [1];  [2];  [3]; ORCiD logo [4]
  1. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Technische Univ., Darmstadt (Germany); Ansa Biotechnologies, Berkeley, CA (United States)
  2. 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)
  3. 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); Shenzhen Inst. for Advanced Technologies (China)
  4. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States)
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), Biological Systems Science Division (SC-23.2); National Science Foundation (NSF)
OSTI Identifier:
1597727
Grant/Contract Number:  
[AC02-05CH11231; NSF EEC 0540879]
Resource Type:
Accepted Manuscript
Journal Name:
Genes
Additional Journal Information:
[ Journal Volume: 11; Journal Issue: 1]; Journal ID: ISSN 2073-4425
Publisher:
MDPI
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; enzymatic DNA synthesis; terminal deoxynucleotidyl transferase; TdT; secondary structures; oligonucleotide synthesis; template-independent polymerase; DNA data storage; thermostability engineering; polymerase cofactors

Citation Formats

Barthel, Sebastian, Palluk, Sebastian, Hillson, Nathan J., Keasling, Jay D., and Arlow, Daniel H. Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3′ Terminal Structures for Enzymatic De Novo DNA Synthesis. United States: N. p., 2020. Web. doi:10.3390/genes11010102.
Barthel, Sebastian, Palluk, Sebastian, Hillson, Nathan J., Keasling, Jay D., & Arlow, Daniel H. Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3′ Terminal Structures for Enzymatic De Novo DNA Synthesis. United States. doi:10.3390/genes11010102.
Barthel, Sebastian, Palluk, Sebastian, Hillson, Nathan J., Keasling, Jay D., and Arlow, Daniel H. Thu . "Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3′ Terminal Structures for Enzymatic De Novo DNA Synthesis". United States. doi:10.3390/genes11010102. https://www.osti.gov/servlets/purl/1597727.
@article{osti_1597727,
title = {Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3′ Terminal Structures for Enzymatic De Novo DNA Synthesis},
author = {Barthel, Sebastian and Palluk, Sebastian and Hillson, Nathan J. and Keasling, Jay D. and Arlow, Daniel H.},
abstractNote = {Enzymatic oligonucleotide synthesis methods based on the template-independent polymerase terminal deoxynucleotidyl transferase (TdT) promise to enable the de novo synthesis of long oligonucleotides under mild, aqueous conditions. Intermediates with a 3' terminal structure (hairpins) will inevitably arise during synthesis, but TdT has poor activity on these structured substrates, limiting its usefulness for oligonucleotide synthesis. Here, we described two parallel efforts to improve the activity of TdT on hairpins: (1) optimization of the concentrations of the divalent cation cofactors and (2) engineering TdT for enhanced thermostability, enabling reactions at elevated temperatures. By combining both of these improvements, we obtained a ~10-fold increase in the elongation rate of a guanine-cytosine hairpin.},
doi = {10.3390/genes11010102},
journal = {Genes},
number = [1],
volume = [11],
place = {United States},
year = {2020},
month = {1}
}

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