Please use this identifier to cite or link to this item: https://doi.org/10.1021/acssynbio.0c00078
DC FieldValue
dc.titleEvolving a Thermostable Terminal Deoxynucleotidyl Transferase
dc.contributor.authorChua, Jasmine Puay Suan
dc.contributor.authorGo, Maybelle Kho
dc.contributor.authorOsothprarop, Trina
dc.contributor.authorMcdonald, Seth
dc.contributor.authorKarabadzhak, Alexander G
dc.contributor.authorYew, Wen Shan
dc.contributor.authorPeisajovich, Sergio
dc.contributor.authorNirantar, Saurabh
dc.date.accessioned2023-05-18T03:02:00Z
dc.date.available2023-05-18T03:02:00Z
dc.date.issued2020-07-17
dc.identifier.citationChua, 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
dc.identifier.issn2161-5063
dc.identifier.urihttps://scholarbank.nus.edu.sg/handle/10635/239484
dc.description.abstractTerminal 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.
dc.language.isoen
dc.publisherAMER CHEMICAL SOC
dc.sourceElements
dc.subjectScience & Technology
dc.subjectLife Sciences & Biomedicine
dc.subjectBiochemical Research Methods
dc.subjectBiochemistry & Molecular Biology
dc.subjectterminal deoxynucleotidyl transferase
dc.subjectTdT
dc.subjectthermostable TdT
dc.subjectthermostability
dc.subjectprotein engineering
dc.subjectmodified nucleotides
dc.subjectIN-SITU
dc.subjectDNA
dc.subjectSTORAGE
dc.subjectMU
dc.typeArticle
dc.date.updated2023-05-18T01:40:42Z
dc.contributor.departmentBIOCHEMISTRY
dc.description.doi10.1021/acssynbio.0c00078
dc.description.sourcetitleACS SYNTHETIC BIOLOGY
dc.description.volume9
dc.description.issue7
dc.description.page1725-1735
dc.published.statePublished
Appears in Collections:Staff Publications
Elements

Show simple item record
Files in This Item:
File Description SizeFormatAccess SettingsVersion 
Evolving a Thermostable Terminal Deoxynucleotidyl Transferase.pdfPublished version3.85 MBAdobe PDF

CLOSED

None

Google ScholarTM

Check

Altmetric


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.