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

Barthel, Sebastian; Palluk, Sebastian; Hillson, Nathan J.; Keasling, Jay D.; Arlow, Daniel H.

doi:10.3390/genes11010102

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:

^[1]; Palluk, Sebastian ^[1]; Hillson, Nathan J. ^[2]; Keasling, Jay D. ^[3];

^[4]

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)
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); Shenzhen Inst. for Advanced Technologies (China)
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:: 2020-01-16

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.

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                    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.  https://doi.org/10.3390/genes11010102

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                    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.  https://doi.org/10.3390/genes11010102.  https://www.osti.gov/servlets/purl/1597727.

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@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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Similar Records

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

Abstract

Citation Formats

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

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

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

Morphological and genetic characterization of the first Isospora species (I. lugensae n. sp.) from a Kerguelen petrel (Lugensa brevirostris) journal, January 2021

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

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

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

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

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

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

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

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

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

Comparison of the Two Murine Deoxynucleotidyltransferase Terminal Isoforms: A 20-AMINO ACID INSERTION IN THE HIGHLY CONSERVED CARBOXYL-TERMINAL REGION MODIFIES THE THERMOSENSITIVITY BUT NOT THE CATALYTIC ACTIVITY journal, June 2000

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

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

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

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

Thermal Conversion of Nonpriming Deoxyribonucleic Acid to Primer journal, October 1959

Multiple roles of divalent cation in the terminal deoxynucleotidyltransferase reaction. journal, October 1990

Interaction of terminal transferase with single-stranded DNA. journal, July 1987

Mutational analysis of residues in the nucleotide binding domain of human terminal deoxynucleotidyl transferase. journal, April 1994

Deoxynucleotide-polymerizing Enzymes of Calf Thymus Gland journal, June 1967

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

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

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

Morphological and genetic characterization of the first Isospora species (I. lugensae n. sp.) from a Kerguelen petrel (Lugensa brevirostris)
journal, January 2021

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

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

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

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

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

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

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

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

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

Comparison of the Two Murine Deoxynucleotidyltransferase Terminal Isoforms: A 20-AMINO ACID INSERTION IN THE HIGHLY CONSERVED CARBOXYL-TERMINAL REGION MODIFIES THE THERMOSENSITIVITY BUT NOT THE CATALYTIC ACTIVITY
journal, June 2000

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

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

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

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

Thermal Conversion of Nonpriming Deoxyribonucleic Acid to Primer
journal, October 1959

Multiple roles of divalent cation in the terminal deoxynucleotidyltransferase reaction.
journal, October 1990

Interaction of terminal transferase with single-stranded DNA.
journal, July 1987

Mutational analysis of residues in the nucleotide binding domain of human terminal deoxynucleotidyl transferase.
journal, April 1994

Deoxynucleotide-polymerizing Enzymes of Calf Thymus Gland
journal, June 1967