Chemo-enzymatic synthesis of site-specific isotopically labeled nucleotides for use in NMR resonance assignment, dynamics and structural characterizations
Identifieur interne : 000969 ( Main/Exploration ); précédent : 000968; suivant : 000970Chemo-enzymatic synthesis of site-specific isotopically labeled nucleotides for use in NMR resonance assignment, dynamics and structural characterizations
Auteurs : Andrew P. Longhini [États-Unis] ; Regan M. Leblanc [États-Unis] ; Owen Becette [États-Unis] ; Carolina Salguero [États-Unis] ; Christoph H. Wunderlich [Autriche] ; Bruce A. Johnson [États-Unis] ; Victoria M. D'Souza [États-Unis] ; Christoph Kreutz [Autriche] ; T. Kwaku Dayie [États-Unis]Source :
- Nucleic Acids Research [ 0305-1048 ] ; 2015.
Descripteurs français
- KwdFr :
- Adénosine triphosphate (synthèse chimique), Bacillus anthracis (), Bacillus anthracis (génétique), Coronavirus humain 229E (), Coronavirus humain 229E (génétique), Creatine kinase (), Creatine kinase (génétique), Guanosine triphosphate (synthèse chimique), Isotopes du carbone, Marquage isotopique (), Nucléotides (synthèse chimique), Pentosyltransferases (), Pentosyltransferases (génétique), Phosphotransferases (Alcohol Group Acceptor) (), Phosphotransferases (Alcohol Group Acceptor) (génétique), Protéines recombinantes (), Protéines recombinantes (génétique), Riborégulateur, Ribose (), Ribose phosphate pyrophosphokinase (), Ribose phosphate pyrophosphokinase (génétique), Spectroscopie par résonance magnétique, Transcription génétique, Éléments de réponse.
- MESH :
- génétique : Bacillus anthracis, Coronavirus humain 229E, Creatine kinase, Pentosyltransferases, Phosphotransferases (Alcohol Group Acceptor), Protéines recombinantes, Ribose phosphate pyrophosphokinase.
- synthèse chimique : Adénosine triphosphate, Guanosine triphosphate, Nucléotides.
- Bacillus anthracis, Coronavirus humain 229E, Creatine kinase, Isotopes du carbone, Marquage isotopique, Pentosyltransferases, Phosphotransferases (Alcohol Group Acceptor), Protéines recombinantes, Riborégulateur, Ribose, Ribose phosphate pyrophosphokinase, Spectroscopie par résonance magnétique, Transcription génétique, Éléments de réponse.
English descriptors
- KwdEn :
- Adenosine Triphosphate (chemical synthesis), Bacillus anthracis (chemistry), Bacillus anthracis (genetics), Carbon Isotopes, Coronavirus 229E, Human (chemistry), Coronavirus 229E, Human (genetics), Creatine Kinase (chemistry), Creatine Kinase (genetics), Guanosine Triphosphate (chemical synthesis), Isotope Labeling (methods), Magnetic Resonance Spectroscopy, Nucleotides (chemical synthesis), Pentosyltransferases (chemistry), Pentosyltransferases (genetics), Phosphotransferases (Alcohol Group Acceptor) (chemistry), Phosphotransferases (Alcohol Group Acceptor) (genetics), Recombinant Proteins (chemistry), Recombinant Proteins (genetics), Response Elements, Ribose (chemistry), Ribose-Phosphate Pyrophosphokinase (chemistry), Ribose-Phosphate Pyrophosphokinase (genetics), Riboswitch, Transcription, Genetic.
- MESH :
- chemical , chemical synthesis : Adenosine Triphosphate, Guanosine Triphosphate, Nucleotides.
- chemistry : Bacillus anthracis, Coronavirus 229E, Human, Creatine Kinase, Pentosyltransferases, Phosphotransferases (Alcohol Group Acceptor), Recombinant Proteins, Ribose, Ribose-Phosphate Pyrophosphokinase.
- genetics : Bacillus anthracis, Coronavirus 229E, Human, Creatine Kinase, Pentosyltransferases, Phosphotransferases (Alcohol Group Acceptor), Recombinant Proteins, Ribose-Phosphate Pyrophosphokinase.
- methods : Isotope Labeling.
- chemical : Carbon Isotopes, Magnetic Resonance Spectroscopy, Response Elements, Riboswitch, Transcription, Genetic.
Abstract
Stable isotope labeling is central to NMR studies of nucleic acids. Development of methods that incorporate labels at specific atomic positions within each nucleotide promises to expand the size range of RNAs that can be studied by NMR. Using recombinantly expressed enzymes and chemically synthesized ribose and nucleobase, we have developed an inexpensive, rapid chemo-enzymatic method to label ATP and GTP site specifically and in high yields of up to 90%. We incorporated these nucleotides into RNAs with sizes ranging from 27 to 59 nucleotides using
Url:
DOI: 10.1093/nar/gkv1333
PubMed: 26657632
PubMed Central: 4824079
Affiliations:
- Autriche, États-Unis
- Maryland, Massachusetts
- Cambridge (Massachusetts), College Park (Maryland)
- Université Harvard, Université du Maryland
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Le document en format XML
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Longhini</name><affiliation wicri:level="4"><nlm:aff id="AFF1">Center for Biomolecular Structure and Organization, Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USAfi</nlm:aff><orgName type="university">Université du Maryland</orgName><country>États-Unis</country><placeName><settlement type="city">College Park (Maryland)</settlement><region type="state">Maryland</region></placeName></affiliation></author><author><name sortKey="Leblanc, Regan M" sort="Leblanc, Regan M" uniqKey="Leblanc R" first="Regan M." last="Leblanc">Regan M. Leblanc</name><affiliation wicri:level="4"><nlm:aff id="AFF1">Center for Biomolecular Structure and Organization, Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USAfi</nlm:aff><orgName type="university">Université du Maryland</orgName><country>États-Unis</country><placeName><settlement type="city">College Park (Maryland)</settlement><region type="state">Maryland</region></placeName></affiliation></author><author><name sortKey="Becette, Owen" sort="Becette, Owen" uniqKey="Becette O" first="Owen" last="Becette">Owen Becette</name><affiliation wicri:level="4"><nlm:aff id="AFF1">Center for Biomolecular Structure and Organization, Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USAfi</nlm:aff><orgName type="university">Université du Maryland</orgName><country>États-Unis</country><placeName><settlement type="city">College Park (Maryland)</settlement><region type="state">Maryland</region></placeName></affiliation></author><author><name sortKey="Salguero, Carolina" sort="Salguero, Carolina" uniqKey="Salguero C" first="Carolina" last="Salguero">Carolina Salguero</name><affiliation wicri:level="4"><nlm:aff id="AFF2">Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138</wicri:regionArea><orgName type="university">Université Harvard</orgName><placeName><settlement type="city">Cambridge (Massachusetts)</settlement><region type="state">Massachusetts</region></placeName></affiliation></author><author><name sortKey="Wunderlich, Christoph H" sort="Wunderlich, Christoph H" uniqKey="Wunderlich C" first="Christoph H." last="Wunderlich">Christoph H. Wunderlich</name><affiliation wicri:level="1"><nlm:aff id="AFF3">Institute of Organic Chemistry and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, 6020 Innsbruck, Austria</nlm:aff><country xml:lang="fr">Autriche</country><wicri:regionArea>Institute of Organic Chemistry and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, 6020 Innsbruck</wicri:regionArea><wicri:noRegion>6020 Innsbruck</wicri:noRegion></affiliation></author><author><name sortKey="Johnson, Bruce A" sort="Johnson, Bruce A" uniqKey="Johnson B" first="Bruce A." last="Johnson">Bruce A. Johnson</name><affiliation wicri:level="1"><nlm:aff id="AFF4">Structural Biology Initiative, CUNY Advanced Science Research Center, 85 St. Nicholas Terrace, New York, NY 10031, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Structural Biology Initiative, CUNY Advanced Science Research Center, 85 St. Nicholas Terrace, New York, NY 10031</wicri:regionArea><wicri:noRegion>NY 10031</wicri:noRegion></affiliation><affiliation wicri:level="1"><nlm:aff id="AFF5">One Moon Scientific, Inc., 839 Grant Avenue, Westfield, NJ 07090-2322, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>One Moon Scientific, Inc., 839 Grant Avenue, Westfield, NJ 07090-2322</wicri:regionArea><wicri:noRegion>NJ 07090-2322</wicri:noRegion></affiliation></author><author><name sortKey="D Souza, Victoria M" sort="D Souza, Victoria M" uniqKey="D Souza V" first="Victoria M." last="D'Souza">Victoria M. D'Souza</name><affiliation wicri:level="4"><nlm:aff id="AFF2">Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138</wicri:regionArea><orgName type="university">Université Harvard</orgName><placeName><settlement type="city">Cambridge (Massachusetts)</settlement><region type="state">Massachusetts</region></placeName></affiliation></author><author><name sortKey="Kreutz, Christoph" sort="Kreutz, Christoph" uniqKey="Kreutz C" first="Christoph" last="Kreutz">Christoph Kreutz</name><affiliation wicri:level="1"><nlm:aff id="AFF3">Institute of Organic Chemistry and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, 6020 Innsbruck, Austria</nlm:aff><country xml:lang="fr">Autriche</country><wicri:regionArea>Institute of Organic Chemistry and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, 6020 Innsbruck</wicri:regionArea><wicri:noRegion>6020 Innsbruck</wicri:noRegion></affiliation></author><author><name sortKey="Dayie, T Kwaku" sort="Dayie, T Kwaku" uniqKey="Dayie T" first="T. Kwaku" last="Dayie">T. Kwaku Dayie</name><affiliation wicri:level="4"><nlm:aff id="AFF1">Center for Biomolecular Structure and Organization, Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USAfi</nlm:aff><orgName type="university">Université du Maryland</orgName><country>États-Unis</country><placeName><settlement type="city">College Park (Maryland)</settlement><region type="state">Maryland</region></placeName></affiliation></author></analytic><series><title level="j">Nucleic Acids Research</title><idno type="ISSN">0305-1048</idno><idno type="eISSN">1362-4962</idno><imprint><date when="2015">2015</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Adenosine Triphosphate (chemical synthesis)</term><term>Bacillus anthracis (chemistry)</term><term>Bacillus anthracis (genetics)</term><term>Carbon Isotopes</term><term>Coronavirus 229E, Human (chemistry)</term><term>Coronavirus 229E, Human (genetics)</term><term>Creatine Kinase (chemistry)</term><term>Creatine Kinase (genetics)</term><term>Guanosine Triphosphate (chemical synthesis)</term><term>Isotope Labeling (methods)</term><term>Magnetic Resonance Spectroscopy</term><term>Nucleotides (chemical synthesis)</term><term>Pentosyltransferases (chemistry)</term><term>Pentosyltransferases (genetics)</term><term>Phosphotransferases (Alcohol Group Acceptor) (chemistry)</term><term>Phosphotransferases (Alcohol Group Acceptor) (genetics)</term><term>Recombinant Proteins (chemistry)</term><term>Recombinant Proteins (genetics)</term><term>Response Elements</term><term>Ribose (chemistry)</term><term>Ribose-Phosphate Pyrophosphokinase (chemistry)</term><term>Ribose-Phosphate Pyrophosphokinase (genetics)</term><term>Riboswitch</term><term>Transcription, Genetic</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Adénosine triphosphate (synthèse chimique)</term><term>Bacillus anthracis ()</term><term>Bacillus anthracis (génétique)</term><term>Coronavirus humain 229E ()</term><term>Coronavirus humain 229E (génétique)</term><term>Creatine kinase ()</term><term>Creatine kinase (génétique)</term><term>Guanosine triphosphate (synthèse chimique)</term><term>Isotopes du carbone</term><term>Marquage isotopique ()</term><term>Nucléotides (synthèse chimique)</term><term>Pentosyltransferases ()</term><term>Pentosyltransferases (génétique)</term><term>Phosphotransferases (Alcohol Group Acceptor) ()</term><term>Phosphotransferases (Alcohol Group Acceptor) (génétique)</term><term>Protéines recombinantes ()</term><term>Protéines recombinantes (génétique)</term><term>Riborégulateur</term><term>Ribose ()</term><term>Ribose phosphate pyrophosphokinase ()</term><term>Ribose phosphate pyrophosphokinase (génétique)</term><term>Spectroscopie par résonance magnétique</term><term>Transcription génétique</term><term>Éléments de réponse</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemical synthesis" xml:lang="en"><term>Adenosine Triphosphate</term><term>Guanosine Triphosphate</term><term>Nucleotides</term></keywords><keywords scheme="MESH" qualifier="chemistry" xml:lang="en"><term>Bacillus anthracis</term><term>Coronavirus 229E, Human</term><term>Creatine Kinase</term><term>Pentosyltransferases</term><term>Phosphotransferases (Alcohol Group Acceptor)</term><term>Recombinant Proteins</term><term>Ribose</term><term>Ribose-Phosphate Pyrophosphokinase</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Bacillus anthracis</term><term>Coronavirus 229E, Human</term><term>Creatine Kinase</term><term>Pentosyltransferases</term><term>Phosphotransferases (Alcohol Group Acceptor)</term><term>Recombinant Proteins</term><term>Ribose-Phosphate Pyrophosphokinase</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Bacillus anthracis</term><term>Coronavirus humain 229E</term><term>Creatine kinase</term><term>Pentosyltransferases</term><term>Phosphotransferases (Alcohol Group Acceptor)</term><term>Protéines recombinantes</term><term>Ribose phosphate pyrophosphokinase</term></keywords><keywords scheme="MESH" qualifier="methods" xml:lang="en"><term>Isotope Labeling</term></keywords><keywords scheme="MESH" qualifier="synthèse chimique" xml:lang="fr"><term>Adénosine triphosphate</term><term>Guanosine triphosphate</term><term>Nucléotides</term></keywords><keywords scheme="MESH" type="chemical" xml:lang="en"><term>Carbon Isotopes</term><term>Magnetic Resonance Spectroscopy</term><term>Response Elements</term><term>Riboswitch</term><term>Transcription, Genetic</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Bacillus anthracis</term><term>Coronavirus humain 229E</term><term>Creatine kinase</term><term>Isotopes du carbone</term><term>Marquage isotopique</term><term>Pentosyltransferases</term><term>Phosphotransferases (Alcohol Group Acceptor)</term><term>Protéines recombinantes</term><term>Riborégulateur</term><term>Ribose</term><term>Ribose phosphate pyrophosphokinase</term><term>Spectroscopie par résonance magnétique</term><term>Transcription génétique</term><term>Éléments de réponse</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Stable isotope labeling is central to NMR studies of nucleic acids. Development of methods that incorporate labels at specific atomic positions within each nucleotide promises to expand the size range of RNAs that can be studied by NMR. Using recombinantly expressed enzymes and chemically synthesized ribose and nucleobase, we have developed an inexpensive, rapid chemo-enzymatic method to label ATP and GTP site specifically and in high yields of up to 90%. We incorporated these nucleotides into RNAs with sizes ranging from 27 to 59 nucleotides using <italic>in vitro</italic> transcription: A-Site (27 nt), the iron responsive elements (29 nt), a fluoride riboswitch from <italic>Bacillus anthracis</italic> (48 nt), and a frame-shifting element from a human corona virus (59 nt). Finally, we showcase the improvement in spectral quality arising from reduced crowding and narrowed linewidths, and accurate analysis of NMR relaxation dispersion (CPMG) and TROSY-based CEST experiments to measure μs-ms time scale motions, and an improved NOESY strategy for resonance assignment. Applications of this selective labeling technology promises to reduce difficulties associated with chemical shift overlap and rapid signal decay that have made it challenging to study the structure and dynamics of large RNAs beyond the 50 nt median size found in the PDB.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Winkler, W" uniqKey="Winkler W">W. Winkler</name></author><author><name sortKey="Nahvi, A" uniqKey="Nahvi A">A. Nahvi</name></author><author><name sortKey="Breaker, R R" uniqKey="Breaker R">R.R. Breaker</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Baker, J L" uniqKey="Baker J">J.L. 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Longhini</name></region><name sortKey="Becette, Owen" sort="Becette, Owen" uniqKey="Becette O" first="Owen" last="Becette">Owen Becette</name><name sortKey="D Souza, Victoria M" sort="D Souza, Victoria M" uniqKey="D Souza V" first="Victoria M." last="D'Souza">Victoria M. D'Souza</name><name sortKey="Dayie, T Kwaku" sort="Dayie, T Kwaku" uniqKey="Dayie T" first="T. Kwaku" last="Dayie">T. Kwaku Dayie</name><name sortKey="Johnson, Bruce A" sort="Johnson, Bruce A" uniqKey="Johnson B" first="Bruce A." last="Johnson">Bruce A. Johnson</name><name sortKey="Johnson, Bruce A" sort="Johnson, Bruce A" uniqKey="Johnson B" first="Bruce A." last="Johnson">Bruce A. Johnson</name><name sortKey="Leblanc, Regan M" sort="Leblanc, Regan M" uniqKey="Leblanc R" first="Regan M." last="Leblanc">Regan M. Leblanc</name><name sortKey="Salguero, Carolina" sort="Salguero, Carolina" uniqKey="Salguero C" first="Carolina" last="Salguero">Carolina Salguero</name></country><country name="Autriche"><noRegion><name sortKey="Wunderlich, Christoph H" sort="Wunderlich, Christoph H" uniqKey="Wunderlich C" first="Christoph H." last="Wunderlich">Christoph H. Wunderlich</name></noRegion><name sortKey="Kreutz, Christoph" sort="Kreutz, Christoph" uniqKey="Kreutz C" first="Christoph" last="Kreutz">Christoph Kreutz</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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