De novo DNA synthesis using polymerase-nucleotide conjugates.
Identifieur interne : 000865 ( PubMed/Corpus ); précédent : 000864; suivant : 000866De novo DNA synthesis using polymerase-nucleotide conjugates.
Auteurs : Sebastian Palluk ; Daniel H. Arlow ; Tristan De Rond ; Sebastian Barthel ; Justine S. Kang ; Rathin Bector ; Hratch M. Baghdassarian ; Alisa N. Truong ; Peter W. Kim ; Anup K. Singh ; Nathan J. Hillson ; Jay D. KeaslingSource :
- Nature biotechnology [ 1546-1696 ] ; 2018.
English descriptors
- KwdEn :
- DNA Nucleotidylexotransferase (chemistry), DNA Nucleotidylexotransferase (genetics), DNA Replication (genetics), DNA-Directed DNA Polymerase (chemistry), DNA-Directed DNA Polymerase (genetics), Nucleosides (chemistry), Nucleosides (genetics), Oligonucleotides (biosynthesis), Oligonucleotides (chemistry), Oligonucleotides (genetics), Organophosphorus Compounds (chemistry).
- MESH :
- chemical , biosynthesis : Oligonucleotides.
- chemical , chemistry : DNA Nucleotidylexotransferase, DNA-Directed DNA Polymerase, Nucleosides, Oligonucleotides, Organophosphorus Compounds.
- chemical , genetics : DNA Nucleotidylexotransferase, DNA-Directed DNA Polymerase, Nucleosides, Oligonucleotides.
- genetics : DNA Replication.
Abstract
Oligonucleotides are almost exclusively synthesized using the nucleoside phosphoramidite method, even though it is limited to the direct synthesis of ∼200 mers and produces hazardous waste. Here, we describe an oligonucleotide synthesis strategy that uses the template-independent polymerase terminal deoxynucleotidyl transferase (TdT). Each TdT molecule is conjugated to a single deoxyribonucleoside triphosphate (dNTP) molecule that it can incorporate into a primer. After incorporation of the tethered dNTP, the 3' end of the primer remains covalently bound to TdT and is inaccessible to other TdT-dNTP molecules. Cleaving the linkage between TdT and the incorporated nucleotide releases the primer and allows subsequent extension. We demonstrate that TdT-dNTP conjugates can quantitatively extend a primer by a single nucleotide in 10-20 s, and that the scheme can be iterated to write a defined sequence. This approach may form the basis of an enzymatic oligonucleotide synthesizer.
DOI: 10.1038/nbt.4173
PubMed: 29912208
Links to Exploration step
pubmed:29912208Le document en format XML
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Arlow</name><affiliation><nlm:affiliation>Joint BioEnergy Institute, Emeryville, California, USA.</nlm:affiliation></affiliation></author><author><name sortKey="De Rond, Tristan" sort="De Rond, Tristan" uniqKey="De Rond T" first="Tristan" last="De Rond">Tristan De Rond</name><affiliation><nlm:affiliation>Joint BioEnergy Institute, Emeryville, California, USA.</nlm:affiliation></affiliation></author><author><name sortKey="Barthel, Sebastian" sort="Barthel, Sebastian" uniqKey="Barthel S" first="Sebastian" last="Barthel">Sebastian Barthel</name><affiliation><nlm:affiliation>Joint BioEnergy Institute, Emeryville, California, USA.</nlm:affiliation></affiliation></author><author><name sortKey="Kang, Justine S" sort="Kang, Justine S" uniqKey="Kang J" first="Justine S" last="Kang">Justine S. Kang</name><affiliation><nlm:affiliation>Joint BioEnergy Institute, Emeryville, California, USA.</nlm:affiliation></affiliation></author><author><name sortKey="Bector, Rathin" sort="Bector, Rathin" uniqKey="Bector R" first="Rathin" last="Bector">Rathin Bector</name><affiliation><nlm:affiliation>Joint BioEnergy Institute, Emeryville, California, USA.</nlm:affiliation></affiliation></author><author><name sortKey="Baghdassarian, Hratch M" sort="Baghdassarian, Hratch M" uniqKey="Baghdassarian H" first="Hratch M" last="Baghdassarian">Hratch M. Baghdassarian</name><affiliation><nlm:affiliation>Joint BioEnergy Institute, Emeryville, California, USA.</nlm:affiliation></affiliation></author><author><name sortKey="Truong, Alisa N" sort="Truong, Alisa N" uniqKey="Truong A" first="Alisa N" last="Truong">Alisa N. Truong</name><affiliation><nlm:affiliation>Joint BioEnergy Institute, Emeryville, California, USA.</nlm:affiliation></affiliation></author><author><name sortKey="Kim, Peter W" sort="Kim, Peter W" uniqKey="Kim P" first="Peter W" last="Kim">Peter W. Kim</name><affiliation><nlm:affiliation>Joint BioEnergy Institute, Emeryville, California, USA.</nlm:affiliation></affiliation></author><author><name sortKey="Singh, Anup K" sort="Singh, Anup K" uniqKey="Singh A" first="Anup K" last="Singh">Anup K. Singh</name><affiliation><nlm:affiliation>Joint BioEnergy Institute, Emeryville, California, USA.</nlm:affiliation></affiliation></author><author><name sortKey="Hillson, Nathan J" sort="Hillson, Nathan J" uniqKey="Hillson N" first="Nathan J" last="Hillson">Nathan J. Hillson</name><affiliation><nlm:affiliation>Joint BioEnergy Institute, Emeryville, California, USA.</nlm:affiliation></affiliation></author><author><name sortKey="Keasling, Jay D" sort="Keasling, Jay D" uniqKey="Keasling J" first="Jay D" last="Keasling">Jay D. Keasling</name><affiliation><nlm:affiliation>Joint BioEnergy Institute, Emeryville, California, USA.</nlm:affiliation></affiliation></author></analytic><series><title level="j">Nature biotechnology</title><idno type="eISSN">1546-1696</idno><imprint><date when="2018" type="published">2018</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>DNA Nucleotidylexotransferase (chemistry)</term><term>DNA Nucleotidylexotransferase (genetics)</term><term>DNA Replication (genetics)</term><term>DNA-Directed DNA Polymerase (chemistry)</term><term>DNA-Directed DNA Polymerase (genetics)</term><term>Nucleosides (chemistry)</term><term>Nucleosides (genetics)</term><term>Oligonucleotides (biosynthesis)</term><term>Oligonucleotides (chemistry)</term><term>Oligonucleotides (genetics)</term><term>Organophosphorus Compounds (chemistry)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="biosynthesis" xml:lang="en"><term>Oligonucleotides</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>DNA Nucleotidylexotransferase</term><term>DNA-Directed DNA Polymerase</term><term>Nucleosides</term><term>Oligonucleotides</term><term>Organophosphorus Compounds</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>DNA Nucleotidylexotransferase</term><term>DNA-Directed DNA Polymerase</term><term>Nucleosides</term><term>Oligonucleotides</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>DNA Replication</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Oligonucleotides are almost exclusively synthesized using the nucleoside phosphoramidite method, even though it is limited to the direct synthesis of ∼200 mers and produces hazardous waste. Here, we describe an oligonucleotide synthesis strategy that uses the template-independent polymerase terminal deoxynucleotidyl transferase (TdT). Each TdT molecule is conjugated to a single deoxyribonucleoside triphosphate (dNTP) molecule that it can incorporate into a primer. After incorporation of the tethered dNTP, the 3' end of the primer remains covalently bound to TdT and is inaccessible to other TdT-dNTP molecules. Cleaving the linkage between TdT and the incorporated nucleotide releases the primer and allows subsequent extension. We demonstrate that TdT-dNTP conjugates can quantitatively extend a primer by a single nucleotide in 10-20 s, and that the scheme can be iterated to write a defined sequence. This approach may form the basis of an enzymatic oligonucleotide synthesizer.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">29912208</PMID><DateCompleted><Year>2018</Year><Month>12</Month><Day>26</Day></DateCompleted><DateRevised><Year>2018</Year><Month>12</Month><Day>26</Day></DateRevised><Article PubModel="Print-Electronic"><Journal><ISSN IssnType="Electronic">1546-1696</ISSN><JournalIssue CitedMedium="Internet"><Volume>36</Volume><Issue>7</Issue><PubDate><Year>2018</Year><Month>08</Month></PubDate></JournalIssue><Title>Nature biotechnology</Title><ISOAbbreviation>Nat. Biotechnol.</ISOAbbreviation></Journal><ArticleTitle>De novo DNA synthesis using polymerase-nucleotide conjugates.</ArticleTitle><Pagination><MedlinePgn>645-650</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1038/nbt.4173</ELocationID><Abstract><AbstractText>Oligonucleotides are almost exclusively synthesized using the nucleoside phosphoramidite method, even though it is limited to the direct synthesis of ∼200 mers and produces hazardous waste. Here, we describe an oligonucleotide synthesis strategy that uses the template-independent polymerase terminal deoxynucleotidyl transferase (TdT). Each TdT molecule is conjugated to a single deoxyribonucleoside triphosphate (dNTP) molecule that it can incorporate into a primer. After incorporation of the tethered dNTP, the 3' end of the primer remains covalently bound to TdT and is inaccessible to other TdT-dNTP molecules. Cleaving the linkage between TdT and the incorporated nucleotide releases the primer and allows subsequent extension. We demonstrate that TdT-dNTP conjugates can quantitatively extend a primer by a single nucleotide in 10-20 s, and that the scheme can be iterated to write a defined sequence. This approach may form the basis of an enzymatic oligonucleotide synthesizer.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Palluk</LastName><ForeName>Sebastian</ForeName><Initials>S</Initials><AffiliationInfo><Affiliation>Joint BioEnergy Institute, Emeryville, California, USA.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Biological Systems and Engineering Division, Lawrence Berkeley National Lab, Berkeley, California, USA.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Department of Biology, Technische Universität Darmstadt, Darmstadt, Germany.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Arlow</LastName><ForeName>Daniel H</ForeName><Initials>DH</Initials><AffiliationInfo><Affiliation>Joint BioEnergy Institute, Emeryville, California, USA.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Biological Systems and Engineering Division, Lawrence Berkeley National Lab, Berkeley, California, USA.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Biophysics Graduate Group, UC Berkeley, Berkeley, California, USA.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Institute for Quantitative Biosciences, UC Berkeley, Berkeley, California, USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>de Rond</LastName><ForeName>Tristan</ForeName><Initials>T</Initials><AffiliationInfo><Affiliation>Joint BioEnergy Institute, Emeryville, California, USA.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Biological Systems and Engineering Division, Lawrence Berkeley National Lab, Berkeley, California, USA.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Department of Chemistry, UC Berkeley, Berkeley, California, USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Barthel</LastName><ForeName>Sebastian</ForeName><Initials>S</Initials><AffiliationInfo><Affiliation>Joint BioEnergy Institute, Emeryville, California, USA.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Biological Systems and Engineering Division, Lawrence Berkeley National Lab, Berkeley, California, USA.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Department of Biology, Technische Universität Darmstadt, Darmstadt, Germany.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Kang</LastName><ForeName>Justine S</ForeName><Initials>JS</Initials><AffiliationInfo><Affiliation>Joint BioEnergy Institute, Emeryville, California, USA.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Biological Systems and Engineering Division, Lawrence Berkeley National Lab, Berkeley, California, USA.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Department of Chemical and Biomolecular Engineering, UC Berkeley, Berkeley, California, USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Bector</LastName><ForeName>Rathin</ForeName><Initials>R</Initials><AffiliationInfo><Affiliation>Joint BioEnergy Institute, Emeryville, California, USA.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Biological Systems and Engineering Division, Lawrence Berkeley National Lab, Berkeley, California, USA.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Department of Chemical and Biomolecular Engineering, UC Berkeley, Berkeley, California, USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Baghdassarian</LastName><ForeName>Hratch M</ForeName><Initials>HM</Initials><AffiliationInfo><Affiliation>Joint BioEnergy Institute, Emeryville, California, USA.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Biological Systems and Engineering Division, Lawrence Berkeley National Lab, Berkeley, California, USA.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Department of Bioengineering UC Berkeley, Berkeley, California, USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Truong</LastName><ForeName>Alisa N</ForeName><Initials>AN</Initials><AffiliationInfo><Affiliation>Joint BioEnergy Institute, Emeryville, California, USA.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Biological Systems and Engineering Division, Lawrence Berkeley National Lab, Berkeley, California, USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Kim</LastName><ForeName>Peter W</ForeName><Initials>PW</Initials><AffiliationInfo><Affiliation>Joint BioEnergy Institute, Emeryville, California, USA.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>CBRN Defense and Energy Technologies, Sandia National Laboratories, Livermore, California, USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Singh</LastName><ForeName>Anup K</ForeName><Initials>AK</Initials><AffiliationInfo><Affiliation>Joint BioEnergy Institute, Emeryville, California, USA.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>CBRN Defense and Energy Technologies, Sandia National Laboratories, Livermore, California, USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Hillson</LastName><ForeName>Nathan J</ForeName><Initials>NJ</Initials><Identifier Source="ORCID">0000-0002-9169-3978</Identifier><AffiliationInfo><Affiliation>Joint BioEnergy Institute, Emeryville, California, USA.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Biological Systems and Engineering Division, Lawrence Berkeley National Lab, Berkeley, California, USA.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>DOE Joint Genome Institute, Walnut Creek, California, USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Keasling</LastName><ForeName>Jay D</ForeName><Initials>JD</Initials><Identifier Source="ORCID">0000-0003-4170-6088</Identifier><AffiliationInfo><Affiliation>Joint BioEnergy Institute, Emeryville, California, USA.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Biological Systems and Engineering Division, Lawrence Berkeley National Lab, Berkeley, California, USA.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Institute for Quantitative Biosciences, UC Berkeley, Berkeley, California, USA.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Department of Chemical and Biomolecular Engineering, UC Berkeley, Berkeley, California, USA.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Department of Bioengineering UC Berkeley, Berkeley, California, USA.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType><PublicationType UI="D013486">Research Support, U.S. Gov't, Non-P.H.S.</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2018</Year><Month>06</Month><Day>18</Day></ArticleDate></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>Nat Biotechnol</MedlineTA><NlmUniqueID>9604648</NlmUniqueID><ISSNLinking>1087-0156</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D009705">Nucleosides</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D009841">Oligonucleotides</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D009943">Organophosphorus Compounds</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="C434331">phosphoramidite</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 2.7.7.31</RegistryNumber><NameOfSubstance UI="D004253">DNA Nucleotidylexotransferase</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 2.7.7.7</RegistryNumber><NameOfSubstance UI="D004259">DNA-Directed DNA Polymerase</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><CommentsCorrectionsList><CommentsCorrections RefType="CommentIn"><RefSource>Nature. 2019 Feb;566(7745):565</RefSource><PMID Version="1">30804572</PMID></CommentsCorrections></CommentsCorrectionsList><MeshHeadingList><MeshHeading><DescriptorName UI="D004253" MajorTopicYN="N">DNA Nucleotidylexotransferase</DescriptorName><QualifierName UI="Q000737" MajorTopicYN="N">chemistry</QualifierName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D004261" MajorTopicYN="N">DNA Replication</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D004259" MajorTopicYN="N">DNA-Directed DNA 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