TU Darmstadt / ULB / TUprints

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. (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,
[Article]

[img]
Preview
Text
genes-11-00102.pdf
Available under: CC BY 4.0 International - Creative Commons, Attribution.

Download (526kB) | Preview
Item Type: Article
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
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: 02 May 2022 12:07
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:
Export:
Actions (login required)
View Item View Item