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
Artikel, Bibliographie
Dies ist die neueste Version dieses Eintrags.
Kurzbeschreibung (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.
| Typ des Eintrags: | Artikel |
|---|---|
| Erschienen: | 2022 |
| Autor(en): | Barthel, Sebastian ; Palluk, Sebastian ; Hillson, Nathan J. ; Keasling, Jay D. ; Arlow, Daniel H. |
| Art des Eintrags: | Bibliographie |
| Titel: | Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3′ Terminal Structures for Enzymatic De Novo DNA Synthesis |
| Sprache: | Englisch |
| Publikationsjahr: | 2022 |
| Verlag: | MDPI |
| Titel der Zeitschrift, Zeitung oder Schriftenreihe: | Genes |
| Jahrgang/Volume einer Zeitschrift: | 11 |
| (Heft-)Nummer: | 1 |
| Kollation: | 9 Seiten |
| DOI: | 10.3390/genes11010102 |
| Zugehörige Links: | |
| Kurzbeschreibung (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. |
| Freie Schlagworte: | enzymatic DNA synthesis, terminal deoxynucleotidyl transferase, TdT, secondary structures, oligonucleotide synthesis, template-independent polymerase, DNA data storage, thermostability engineering, polymerase cofactors |
| Sachgruppe der Dewey Dezimalklassifikatin (DDC): | 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie |
| Fachbereich(e)/-gebiet(e): | 10 Fachbereich Biologie 10 Fachbereich Biologie > Molecular Genetics |
| Hinterlegungsdatum: | 02 Aug 2024 12:37 |
| Letzte Änderung: | 02 Aug 2024 12:37 |
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Verfügbare Versionen dieses Eintrags
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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) [Gegenwärtig angezeigt]
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