Chemo-enzymatic synthesis of site-specific isotopically labeled nucleotides for use in NMR resonance assignment, dynamics and structural characterizations
Identifieur interne : 000046 ( Pmc/Curation ); précédent : 000045; suivant : 000047Chemo-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.
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
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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="1"><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></affiliation></author><author><name sortKey="Wunderlich, Christoph H" sort="Wunderlich, Christoph H" uniqKey="Wunderlich C" first="Christoph H." last="Wunderlich">Christoph H. 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D'Souza</name><affiliation wicri:level="1"><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></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></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></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">26657632</idno><idno type="pmc">4824079</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4824079</idno><idno type="RBID">PMC:4824079</idno><idno type="doi">10.1093/nar/gkv1333</idno><date when="2015">2015</date><idno type="wicri:Area/Pmc/Corpus">000046</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000046</idno><idno type="wicri:Area/Pmc/Curation">000046</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Curation">000046</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Chemo-enzymatic synthesis of site-specific isotopically labeled nucleotides for use in NMR resonance assignment, dynamics and structural characterizations</title><author><name sortKey="Longhini, Andrew P" sort="Longhini, Andrew P" uniqKey="Longhini A" first="Andrew P." last="Longhini">Andrew P. 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="1"><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></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></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></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></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="1"><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></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></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></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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<front><journal-meta><journal-id journal-id-type="nlm-ta">Nucleic Acids Res</journal-id><journal-id journal-id-type="iso-abbrev">Nucleic Acids Res</journal-id><journal-id journal-id-type="hwp">nar</journal-id><journal-id journal-id-type="publisher-id">nar</journal-id><journal-title-group><journal-title>Nucleic Acids Research</journal-title></journal-title-group><issn pub-type="ppub">0305-1048</issn><issn pub-type="epub">1362-4962</issn><publisher><publisher-name>Oxford University Press</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">26657632</article-id><article-id pub-id-type="pmc">4824079</article-id><article-id pub-id-type="doi">10.1093/nar/gkv1333</article-id><article-categories><subj-group subj-group-type="hwp-journal-coll"><subject>13</subject><subject>14</subject></subj-group><subj-group subj-group-type="heading"><subject>Methods Online</subject></subj-group></article-categories><title-group><article-title>Chemo-enzymatic synthesis of site-specific isotopically labeled nucleotides for use in NMR resonance assignment, dynamics and structural characterizations</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Longhini</surname><given-names>Andrew P.</given-names></name><xref ref-type="aff" rid="AFF1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>LeBlanc</surname><given-names>Regan M.</given-names></name><xref ref-type="aff" rid="AFF1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Becette</surname><given-names>Owen</given-names></name><xref ref-type="aff" rid="AFF1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Salguero</surname><given-names>Carolina</given-names></name><xref ref-type="aff" rid="AFF2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Wunderlich</surname><given-names>Christoph H.</given-names></name><xref ref-type="aff" rid="AFF3"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>Johnson</surname><given-names>Bruce A.</given-names></name><xref ref-type="aff" rid="AFF4"><sup>4</sup></xref><xref ref-type="aff" rid="AFF5"><sup>5</sup></xref></contrib><contrib contrib-type="author"><name><surname>D'Souza</surname><given-names>Victoria M.</given-names></name><xref ref-type="aff" rid="AFF2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Kreutz</surname><given-names>Christoph</given-names></name><xref ref-type="aff" rid="AFF3"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>Dayie</surname><given-names>T. Kwaku</given-names></name><xref ref-type="aff" rid="AFF1"><sup>1</sup></xref><xref ref-type="corresp" rid="COR1">*</xref></contrib><aff id="AFF1"><label>1</label>Center for Biomolecular Structure and Organization, Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USAfi</aff><aff id="AFF2"><label>2</label>Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA</aff><aff id="AFF3"><label>3</label>Institute of Organic Chemistry and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, 6020 Innsbruck, Austria</aff><aff id="AFF4"><label>4</label>Structural Biology Initiative, CUNY Advanced Science Research Center, 85 St. Nicholas Terrace, New York, NY 10031, USA</aff><aff id="AFF5"><label>5</label>One Moon Scientific, Inc., 839 Grant Avenue, Westfield, NJ 07090-2322, USA</aff></contrib-group><author-notes><corresp id="COR1"><label>*</label>To whom correspondence should be addressed. Tel: +1 301 405 3165; Fax: +1 301 314 0386; Email: <email>dayie@umd.edu</email></corresp></author-notes><pub-date pub-type="ppub"><day>07</day><month>4</month><year>2016</year></pub-date><pub-date pub-type="epub"><day>10</day><month>12</month><year>2015</year></pub-date><pub-date pub-type="pmc-release"><day>10</day><month>12</month><year>2015</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on the
<volume>44</volume><issue>6</issue><fpage>e52</fpage><lpage>e52</lpage><history><date date-type="accepted"><day>16</day><month>11</month><year>2015</year></date><date date-type="rev-recd"><day>14</day><month>11</month><year>2015</year></date><date date-type="received"><day>15</day><month>6</month><year>2015</year></date></history><permissions><copyright-statement>© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.</copyright-statement><copyright-year>2016</copyright-year><license license-type="creative-commons" xlink:href="http://creativecommons.org/licenses/by-nc/4.0/"><license-p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by-nc/4.0/">http://creativecommons.org/licenses/by-nc/4.0/</ext-link>), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact <email>journals.permissions@oup.com</email></license-p></license></permissions><self-uri xlink:title="pdf" xlink:href="gkv1333.pdf"></self-uri><abstract><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></abstract><counts><page-count count="10"></page-count></counts><custom-meta-group><custom-meta><meta-name>cover-date</meta-name><meta-value>07 April 2016</meta-value></custom-meta></custom-meta-group></article-meta></front></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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