De novo DNA synthesis using polymerase-nucleotide conjugates.
Identifieur interne : 000A14 ( Main/Exploration ); précédent : 000A13; suivant : 000A15De novo DNA synthesis using polymerase-nucleotide conjugates.
Auteurs : Sebastian Palluk [États-Unis] ; Daniel H. Arlow [États-Unis] ; Tristan De Rond [États-Unis] ; Sebastian Barthel [États-Unis] ; Justine S. Kang [États-Unis] ; Rathin Bector [États-Unis] ; Hratch M. Baghdassarian [États-Unis] ; Alisa N. Truong [États-Unis] ; Peter W. Kim [États-Unis] ; Anup K. Singh [États-Unis] ; Nathan J. Hillson [États-Unis] ; Jay D. Keasling [États-Unis]Source :
- Nature biotechnology [ 1546-1696 ] ; 2018.
Descripteurs français
- KwdFr :
- Composés organiques du phosphore (), DNA nucleotidylexotransferase (), DNA nucleotidylexotransferase (génétique), DNA-directed DNA polymerase (), DNA-directed DNA polymerase (génétique), Nucléosides (), Nucléosides (génétique), Oligonucléotides (), Oligonucléotides (biosynthèse), Oligonucléotides (génétique), Réplication de l'ADN (génétique).
- MESH :
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
Affiliations:
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Arlow</name><affiliation wicri:level="2"><nlm:affiliation>Joint BioEnergy Institute, Emeryville, California, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Joint BioEnergy Institute, Emeryville, California</wicri:regionArea><placeName><region type="state">Californie</region></placeName></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 wicri:level="2"><nlm:affiliation>Joint BioEnergy Institute, Emeryville, California, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Joint BioEnergy Institute, Emeryville, California</wicri:regionArea><placeName><region type="state">Californie</region></placeName></affiliation></author><author><name sortKey="Barthel, Sebastian" sort="Barthel, Sebastian" uniqKey="Barthel S" first="Sebastian" last="Barthel">Sebastian Barthel</name><affiliation wicri:level="2"><nlm:affiliation>Joint BioEnergy Institute, Emeryville, California, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Joint BioEnergy Institute, Emeryville, California</wicri:regionArea><placeName><region type="state">Californie</region></placeName></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 wicri:level="2"><nlm:affiliation>Joint BioEnergy Institute, Emeryville, California, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Joint BioEnergy Institute, Emeryville, California</wicri:regionArea><placeName><region type="state">Californie</region></placeName></affiliation></author><author><name sortKey="Bector, Rathin" sort="Bector, Rathin" uniqKey="Bector R" first="Rathin" last="Bector">Rathin Bector</name><affiliation wicri:level="2"><nlm:affiliation>Joint BioEnergy Institute, Emeryville, California, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Joint BioEnergy Institute, Emeryville, California</wicri:regionArea><placeName><region type="state">Californie</region></placeName></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 wicri:level="2"><nlm:affiliation>Joint BioEnergy Institute, Emeryville, California, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Joint BioEnergy Institute, Emeryville, California</wicri:regionArea><placeName><region type="state">Californie</region></placeName></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 wicri:level="2"><nlm:affiliation>Joint BioEnergy Institute, Emeryville, California, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Joint BioEnergy Institute, Emeryville, California</wicri:regionArea><placeName><region type="state">Californie</region></placeName></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 wicri:level="2"><nlm:affiliation>Joint BioEnergy Institute, Emeryville, California, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Joint BioEnergy Institute, Emeryville, California</wicri:regionArea><placeName><region type="state">Californie</region></placeName></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 wicri:level="2"><nlm:affiliation>Joint BioEnergy Institute, Emeryville, California, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Joint BioEnergy Institute, Emeryville, California</wicri:regionArea><placeName><region type="state">Californie</region></placeName></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 wicri:level="2"><nlm:affiliation>Joint BioEnergy Institute, Emeryville, California, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Joint BioEnergy Institute, Emeryville, California</wicri:regionArea><placeName><region type="state">Californie</region></placeName></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 wicri:level="2"><nlm:affiliation>Joint BioEnergy Institute, Emeryville, California, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Joint BioEnergy Institute, Emeryville, California</wicri:regionArea><placeName><region type="state">Californie</region></placeName></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="KwdFr" xml:lang="fr"><term>Composés organiques du phosphore ()</term><term>DNA nucleotidylexotransferase ()</term><term>DNA nucleotidylexotransferase (génétique)</term><term>DNA-directed DNA polymerase ()</term><term>DNA-directed DNA polymerase (génétique)</term><term>Nucléosides ()</term><term>Nucléosides (génétique)</term><term>Oligonucléotides ()</term><term>Oligonucléotides (biosynthèse)</term><term>Oligonucléotides (génétique)</term><term>Réplication de l'ADN (génétique)</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="biosynthèse" xml:lang="fr"><term>Oligonucléotides</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>DNA Replication</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>DNA nucleotidylexotransferase</term><term>DNA-directed DNA polymerase</term><term>Nucléosides</term><term>Oligonucléotides</term><term>Réplication de l'ADN</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Composés organiques du phosphore</term><term>DNA nucleotidylexotransferase</term><term>DNA-directed DNA polymerase</term><term>Nucléosides</term><term>Oligonucléotides</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><affiliations><list><country><li>États-Unis</li></country><region><li>Californie</li></region></list><tree><country name="États-Unis"><region name="Californie"><name sortKey="Palluk, Sebastian" sort="Palluk, Sebastian" uniqKey="Palluk S" first="Sebastian" last="Palluk">Sebastian Palluk</name></region><name sortKey="Arlow, Daniel H" sort="Arlow, Daniel H" uniqKey="Arlow D" first="Daniel H" last="Arlow">Daniel H. Arlow</name><name sortKey="Baghdassarian, Hratch M" sort="Baghdassarian, Hratch M" uniqKey="Baghdassarian H" first="Hratch M" last="Baghdassarian">Hratch M. Baghdassarian</name><name sortKey="Barthel, Sebastian" sort="Barthel, Sebastian" uniqKey="Barthel S" first="Sebastian" last="Barthel">Sebastian Barthel</name><name sortKey="Bector, Rathin" sort="Bector, Rathin" uniqKey="Bector R" first="Rathin" last="Bector">Rathin Bector</name><name sortKey="De Rond, Tristan" sort="De Rond, Tristan" uniqKey="De Rond T" first="Tristan" last="De Rond">Tristan De Rond</name><name sortKey="Hillson, Nathan J" sort="Hillson, Nathan J" uniqKey="Hillson N" first="Nathan J" last="Hillson">Nathan J. Hillson</name><name sortKey="Kang, Justine S" sort="Kang, Justine S" uniqKey="Kang J" first="Justine S" last="Kang">Justine S. Kang</name><name sortKey="Keasling, Jay D" sort="Keasling, Jay D" uniqKey="Keasling J" first="Jay D" last="Keasling">Jay D. Keasling</name><name sortKey="Kim, Peter W" sort="Kim, Peter W" uniqKey="Kim P" first="Peter W" last="Kim">Peter W. Kim</name><name sortKey="Singh, Anup K" sort="Singh, Anup K" uniqKey="Singh A" first="Anup K" last="Singh">Anup K. Singh</name><name sortKey="Truong, Alisa N" sort="Truong, Alisa N" uniqKey="Truong A" first="Alisa N" last="Truong">Alisa N. Truong</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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