TU Darmstadt / ULB / TUbiblio

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.
In: Genes, 2022, 11 (1)
doi: 10.26083/tuprints-00016115
Artikel, Zweitveröffentlichung, Verlagsversion

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: Zweitveröffentlichung
Titel: Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3′ Terminal Structures for Enzymatic De Novo DNA Synthesis
Sprache: Englisch
Publikationsjahr: 2022
Publikationsdatum der Erstveröffentlichung: 2022
Verlag: MDPI
Titel der Zeitschrift, Zeitung oder Schriftenreihe: Genes
Jahrgang/Volume einer Zeitschrift: 11
(Heft-)Nummer: 1
Kollation: 9 Seiten
DOI: 10.26083/tuprints-00016115
URL / URN: https://tuprints.ulb.tu-darmstadt.de/16115
Zugehörige Links:
Herkunft: Zweitveröffentlichung
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
Status: Verlagsversion
URN: urn:nbn:de:tuda-tuprints-161159
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: 09 Feb 2022 15:26
Letzte Änderung: 10 Feb 2022 06:43
PPN:
Zugehörige Links:
Export:
Suche nach Titel in: TUfind oder in Google
Frage zum Eintrag Frage zum Eintrag

Optionen (nur für Redakteure)
Redaktionelle Details anzeigen Redaktionelle Details anzeigen