Barthel, Sebastian ; Palluk, Sebastian ; Hillson, Nathan J. ; Keasling, Jay D. ; Arlow, Daniel H. (2022):
Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3′ Terminal Structures for Enzymatic De Novo DNA Synthesis. (Publisher's Version)
In: Genes, 11 (1), MDPI, e-ISSN 2073-4425,
DOI: 10.26083/tuprints-00016115,
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Item Type: | Article |
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Origin: | Secondary publication DeepGreen |
Status: | Publisher's Version |
Title: | Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3′ Terminal Structures for Enzymatic De Novo DNA Synthesis |
Language: | English |
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 30 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. |
Journal or Publication Title: | Genes |
Volume of the journal: | 11 |
Issue Number: | 1 |
Place of Publication: | Darmstadt |
Publisher: | MDPI |
Collation: | 9 Seiten |
Uncontrolled Keywords: | enzymatic DNA synthesis, terminal deoxynucleotidyl transferase, TdT, secondary structures, oligonucleotide synthesis, template-independent polymerase, DNA data storage, thermostability engineering, polymerase cofactors |
Classification DDC: | 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie |
Divisions: | 10 Department of Biology > Molecular Genetics |
Date Deposited: | 09 Feb 2022 15:26 |
Last Modified: | 07 Mar 2023 07:33 |
DOI: | 10.26083/tuprints-00016115 |
Corresponding Links: | |
URN: | urn:nbn:de:tuda-tuprints-161159 |
SWORD Depositor: | Deep Green |
URI: | https://tuprints.ulb.tu-darmstadt.de/id/eprint/16115 |
PPN: | 505568047 |
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