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

by Sebastian Barthel 1,2,3,†, Sebastian Palluk 1,2,3,†, Nathan J. Hillson 1,2,4, Jay D. Keasling 1,2,5,6,7,8,9 and Daniel H. Arlow 1,2,5,10,*,†
1
Joint BioEnergy Institute, Emeryville, CA 94608, USA
2
Biological Systems and Engineering Division, Lawrence Berkeley National Lab, Berkeley, CA 94720, USA
3
Department of Biology, Technische Universität Darmstadt, 64287 Darmstadt, Germany
4
DOE Joint Genome Institute, Walnut Creek, CA 94598, USA
5
Institute for Quantitative Biosciences, UC Berkeley, Berkeley, CA 94720, USA
6
Department of Chemical and Biomolecular Engineering, UC Berkeley, Berkeley, CA 94720, USA
7
Department of Bioengineering UC Berkeley, Berkeley, CA 94720, USA
8
Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2970 Hørsholm, Denmark
9
Center for Synthetic Biochemistry, Institute for Synthetic Biology, Shenzhen Institutes for Advanced Technologies, Shenzhen 518055, China
10
Biophysics Graduate Group, UC Berkeley, Berkeley, CA 94720, USA
*
Author to whom correspondence should be addressed.
Current address: Ansa Biotechnologies, Berkeley, CA 94170, USA.
Genes 2020, 11(1), 102; https://doi.org/10.3390/genes11010102
Received: 8 December 2019 / Revised: 31 December 2019 / Accepted: 7 January 2020 / Published: 16 January 2020
(This article belongs to the Special Issue eGenetics)
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. View Full-Text
Keywords: enzymatic DNA synthesis; terminal deoxynucleotidyl transferase; TdT; secondary structures; oligonucleotide synthesis; template-independent polymerase; DNA data storage; thermostability engineering; polymerase cofactors enzymatic DNA synthesis; terminal deoxynucleotidyl transferase; TdT; secondary structures; oligonucleotide synthesis; template-independent polymerase; DNA data storage; thermostability engineering; polymerase cofactors
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Barthel, S.; Palluk, S.; Hillson, N.J.; Keasling, J.D.; Arlow, D.H. Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3′ Terminal Structures for Enzymatic De Novo DNA Synthesis. Genes 2020, 11, 102.

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