Sequence Preference and Initiator Promiscuity for De Novo DNA Synthesis by Terminal Deoxynucleotidyl Transferase
- Author(s)
- Erika Schaudy, Jory Lietard, Mark M. Somoza
- Abstract
The untemplated activity of terminal deoxynucleotidyl transferase (TdT) represents its most appealing feature. Its use is well established in applications aiming for extension of a DNA initiator strand, but a more recent focus points to its potential in enzymatic de novo synthesis of DNA. Whereas its low substrate specificity for nucleoside triphosphates has been studied extensively, here we interrogate how the activity of TdT is modulated by the nature of the initiating strands, in particular their length, chemistry, and nucleotide composition. Investigation of full permutational libraries of mono- to pentamers of D-DNA, L-DNA, and 2'O-methyl-RNA of differing directionality immobilized to glass surfaces, and generated via photolithographic in situ synthesis, shows that the efficiency of extension strongly depends on the nucleobase sequence. We also show TdT being catalytically active on a non-nucleosidic substrate, hexaethylene glycol. These results offer new perspectives on constraints and strategies for de novo synthesis of DNA using TdT regarding the requirements for initiation of enzymatic generation of DNA.
- Organisation(s)
- Department of Inorganic Chemistry
- External organisation(s)
- Technische Universität München
- Journal
- ACS Synthetic Biology
- Volume
- 10
- Pages
- 1750-1760
- No. of pages
- 11
- ISSN
- 2161-5063
- DOI
- https://doi.org/10.1021/acssynbio.1c00142
- Publication date
- 07-2021
- Peer reviewed
- Yes
- Austrian Fields of Science 2012
- 106002 Biochemistry, 106023 Molecular biology
- Keywords
- ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology (miscellaneous), Biomedical Engineering
- Portal url
- https://ucrisportal.univie.ac.at/en/publications/b77bdebd-3b5c-4e9f-a864-78fb9f600b0c