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https://doi.org/10.1021/acssynbio.0c00078
Title: | Evolving a Thermostable Terminal Deoxynucleotidyl Transferase | Authors: | Chua, Jasmine Puay Suan Go, Maybelle Kho Osothprarop, Trina Mcdonald, Seth Karabadzhak, Alexander G Yew, Wen Shan Peisajovich, Sergio Nirantar, Saurabh |
Keywords: | Science & Technology Life Sciences & Biomedicine Biochemical Research Methods Biochemistry & Molecular Biology terminal deoxynucleotidyl transferase TdT thermostable TdT thermostability protein engineering modified nucleotides IN-SITU DNA STORAGE MU |
Issue Date: | 17-Jul-2020 | Publisher: | AMER CHEMICAL SOC | Citation: | Chua, Jasmine Puay Suan, Go, Maybelle Kho, Osothprarop, Trina, Mcdonald, Seth, Karabadzhak, Alexander G, Yew, Wen Shan, Peisajovich, Sergio, Nirantar, Saurabh (2020-07-17). Evolving a Thermostable Terminal Deoxynucleotidyl Transferase. ACS SYNTHETIC BIOLOGY 9 (7) : 1725-1735. ScholarBank@NUS Repository. https://doi.org/10.1021/acssynbio.0c00078 | Abstract: | Terminal deoxynucleotidyl transferase (TdT) catalyzes template free incorporation of arbitrary nucleotides onto single-stranded DNA. Due to this unique feature, TdT is widely used in biotechnology and clinical applications. One particularly tantalizing use is the synthesis of long de novo DNA molecules by TdT-mediated iterative incorporation of a 3′ reversibly blocked nucleotide, followed by deblocking. However, wild-type (WT) TdT is not optimized for the incorporation of 3′ modified nucleotides, and TdT engineering is hampered by the fact that TdT is marginally stable and only present in mesophilic organisms. We sought to first evolve a thermostable TdT variant to serve as backbone for subsequent evolution to enable efficient incorporation of 3′-modified nucleotides. A thermostable variant would be a good starting point for such an effort, as evolution to incorporate bulky modified nucleotides generally results in lowered stability. In addition, a thermostable TdT would also be useful when blunt dsDNA is a substrate as higher temperature could be used to melt dsDNA. Here, we developed an assay to identify thermostable TdT variants. After screening about 10 000 TdT mutants, we identified a variant, named TdT3-2, that is 10 °C more thermostable than WT TdT, while preserving the catalytic properties of the WT enzyme. | Source Title: | ACS SYNTHETIC BIOLOGY | URI: | https://scholarbank.nus.edu.sg/handle/10635/239484 | ISSN: | 2161-5063 | DOI: | 10.1021/acssynbio.0c00078 |
Appears in Collections: | Staff Publications Elements |
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Evolving a Thermostable Terminal Deoxynucleotidyl Transferase.pdf | Published version | 3.85 MB | Adobe PDF | CLOSED | None |
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