Template-Independent Enzymatic Oligonucleotide Synthesis (TiEOS): Its History, Prospects, and Challenges
Open accessAbstract
There is a growing demand for sustainable methods in research and development, where instead of hazardous chemicals, an aqueous medium is chosen to perform biological reactions. In this Perspective, we examine the history and current methodology of using enzymes to generate artificial single-stranded DNA. By using traditional solid-phase phosphoramidite chemistry as a metric, we also explore criteria for the method of template-independent enzymatic oligonucleotide synthesis (TiEOS). As its key component, we delve into the biology of one of the most enigmatic enzymes, terminal deoxynucleotidyl transferase (TdT). As TdT is found to exponentially increase antigen receptor diversity in the vertebrate immune system by adding nucleotides in a template-free manner, researchers have exploited this function as an alternative to the phosphoramidite synthesis method. Though TdT is currently the preferred enzyme for TiEOS, its random nucleotide incorporation presents a barrier in synthesis automation. Taking a closer look at the TiEOS cycle, particularly the coupling step, we find it is comprised of additions > n+1 and deletions. By tapping into the physical and biochemical properties of TdT, we strive to further elucidate its mercurial behavior and offer ways to better optimize TiEOS for production-grade oligonucleotide synthesis.
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The costs of enzymatic synthesis would be 1 order of magnitude lower than the phosphoramidite technique if the TdT was recycled.
“Thus, the costs of the enzymatic synthesis would be 1 order of magnitude lower than the phosphoramidite technique if the TdT was recycled.”
From Emerging Approaches to DNA Data Storage: Challenges and Prospects (2022) · cites this paper as [100]
The total costs of synthesizing 1000 strands of 1000 nucleotide length would be 136 USD with recycled TdT, 2700 USD by phosphoramidite technique, and 136,000 USD if a fresh stock of TdT was introduced at every cycle.
“The total costs of synthesizing 1000 strands of 1000 nucleotide length would be 136 USD with recycled TdT, 2700 USD by phosphoramidite technique, and 136 000 USD if a fresh stock of TdT was introduced at every cycle.”
From Emerging Approaches to DNA Data Storage: Challenges and Prospects (2022) · cites this paper as [100]
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- Authors
- Jensen, M. A., & Davis, R. W.
- Affiliations
- Stanford University (US)
- Corresponding author
- Jensen, Michael A.
- Venue
- Biochemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2018-03-13
- Volume
- 57
- Issue
- 12
- Pages
- 1821-1832
- ISSN
- 0006-2960 (ISSN-L)
- 1520-4995
- PMC
- PMC7995564
- OpenAlex
- W2793289833
- Cited by
- 106
- References
- 105
- Citations by year
- 2026: 15 · 2025: 23 · 2024: 26 · 2023: 14 · 2022: 13 · 2021: 4 · 2020: 5 · 2019: 3 · 2018: 3
- FWCI
- 0.37
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Biochemistry (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Human Genome Research Institute (2P01HG000205-24)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Keywords
- terminal deoxynucleotidyl transferase, single-stranded DNA synthesis, phosphoramidite chemistry, antigen receptor diversity, aqueous synthesis
- MeSH terms
- DNA Nucleotidylexotransferase, Oligonucleotides, History, 20th Century, History, 21st Century
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