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.
In: Genes, 11 (1)
doi: 10.3390/genes11010102
Article, Bibliographie
This is the latest version of this item.
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.
| Item Type: | Article |
|---|---|
| Erschienen: | 2022 |
| Creators: | Barthel, Sebastian ; Palluk, Sebastian ; Hillson, Nathan J. ; Keasling, Jay D. ; Arlow, Daniel H. |
| Type of entry: | Bibliographie |
| Title: | Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3′ Terminal Structures for Enzymatic De Novo DNA Synthesis |
| Language: | English |
| Date: | 2022 |
| Publisher: | MDPI |
| Journal or Publication Title: | Genes |
| Volume of the journal: | 11 |
| Issue Number: | 1 |
| Collation: | 9 Seiten |
| DOI: | 10.3390/genes11010102 |
| Corresponding Links: | |
| 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. |
| 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 Science and mathematics > 570 Life sciences, biology |
| Divisions: | 10 Department of Biology 10 Department of Biology > Molecular Genetics |
| Date Deposited: | 02 Aug 2024 12:37 |
| Last Modified: | 02 Aug 2024 12:37 |
| PPN: | |
| Export: | |
| Suche nach Titel in: | TUfind oder in Google |
Available Versions of this Item
-
Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3′ Terminal Structures for Enzymatic De Novo DNA Synthesis. (deposited 09 Feb 2022 15:26)
- Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3′ Terminal Structures for Enzymatic De Novo DNA Synthesis. (deposited 02 Aug 2024 12:37) [Currently Displayed]
 |
Send an inquiry |
Options (only for editors)
 |
Show editorial Details |
Drucken
Impressum