skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

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]
+ Show Author Affiliations
  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.
Copy to clipboard
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:https://doi.org/10.3390/genes11010102
Copy to clipboard
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:https://doi.org/10.3390/genes11010102. https://www.osti.gov/servlets/purl/1597727.
Copy to clipboard
@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}
}
Copy to clipboard
Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
DOI:  https://doi.org/10.3390/genes11010102
Copyright Statement
Other availability
Search WorldCat Search WorldCat to find libraries that may hold this journal
Save / Share:
Export Metadata
Save to My Library
You must Sign In or Create an Account in order to save documents to your library.

Works referenced in this record:

Terminal labeling and addition of homopolymer tracts to duplex DNA fragments by terminal deoxynucleotidyl transferase
journal, April 1976

  • Roychoudhury, R.; Jay, E.; Wu, R.
  • Nucleic Acids Research, Vol. 3, Issue 4
  • DOI: 10.1093/nar/3.4.863

Mfold web server for nucleic acid folding and hybridization prediction
journal, July 2003

De novo DNA synthesis using polymerase-nucleotide conjugates
journal, June 2018

  • Palluk, Sebastian; Arlow, Daniel H.; de Rond, Tristan
  • Nature Biotechnology, Vol. 36, Issue 7
  • DOI: 10.1038/nbt.4173

FireProt: web server for automated design of thermostable proteins
journal, April 2017

  • Musil, Milos; Stourac, Jan; Bendl, Jaroslav
  • Nucleic Acids Research, Vol. 45, Issue W1
  • DOI: 10.1093/nar/gkx285

Terminator-free template-independent enzymatic DNA synthesis for digital information storage
journal, June 2019

FireProt: Energy- and Evolution-Based Computational Design of Thermostable Multiple-Point Mutants
journal, November 2015

Photo-cleavable nucleotides for primer free enzyme mediated DNA synthesis
journal, January 2016

  • Mathews, Anu Stella; Yang, Haikang; Montemagno, Carlo
  • Organic & Biomolecular Chemistry, Vol. 14, Issue 35
  • DOI: 10.1039/C6OB01371F

Metal Ions as Stabilizers or Destabilizers of the Deoxyribonucleic Acid Structure
journal, May 1962

The FoldX web server: an online force field
journal, July 2005

  • Schymkowitz, J.; Borg, J.; Stricher, F.
  • Nucleic Acids Research, Vol. 33, Issue Web Server
  • DOI: 10.1093/nar/gki387

Predicting Stability of DNA Duplexes in Solutions Containing Magnesium and Monovalent Cations
journal, May 2008

  • Owczarzy, Richard; Moreira, Bernardo G.; You, Yong
  • Biochemistry, Vol. 47, Issue 19
  • DOI: 10.1021/bi702363u

Conferring a template-dependent polymerase activity to terminal deoxynucleotidyltransferase by mutations in the Loop1 region
journal, June 2009

  • Romain, Félix; Barbosa, Isabelle; Gouge, Jérôme
  • Nucleic Acids Research, Vol. 37, Issue 14
  • DOI: 10.1093/nar/gkp460

Crystal structures of a template-independent DNA polymerase: murine terminal deoxynucleotidyltransferase
journal, February 2002

Structures of Intermediates along the Catalytic Cycle of Terminal Deoxynucleotidyltransferase: Dynamical Aspects of the Two-Metal Ion Mechanism
journal, November 2013

  • Gouge, Jérôme; Rosario, Sandrine; Romain, Félix
  • Journal of Molecular Biology, Vol. 425, Issue 22
  • DOI: 10.1016/j.jmb.2013.07.009

Influence of Magnesium Ions on Spontaneous Opening of DNA Base Pairs
journal, June 2008

  • Every, Alicia E.; Russu, Irina M.
  • The Journal of Physical Chemistry B, Vol. 112, Issue 25
  • DOI: 10.1021/jp8005876

The Chemical Synthesis of DNA/RNA: Our Gift to Science
journal, December 2012

Analysis of Protein Stability and Ligand Interactions by Thermal Shift Assay
journal, February 2015

Next-Generation Digital Information Storage in DNA
journal, August 2012

Towards synthetic biological approaches to resource utilization on space missions
journal, January 2015

  • Menezes, Amor A.; Cumbers, John; Hogan, John A.
  • Journal of The Royal Society Interface, Vol. 12, Issue 102
  • DOI: 10.1098/rsif.2014.0715
    [ × clear filter / sort ]

    Similar Records in DOE PAGES and OSTI.GOV collections: