Detection of nucleic acid hybridization by nonradiative fluorescence resonance energy transfer.
Identifieur interne : 002A50 ( PubMed/Corpus ); précédent : 002A49; suivant : 002A51Detection of nucleic acid hybridization by nonradiative fluorescence resonance energy transfer.
Auteurs : R A Cardullo ; S. Agrawal ; C. Flores ; P C Zamecnik ; D E WolfSource :
- Proceedings of the National Academy of Sciences of the United States of America [ 0027-8424 ] ; 1988.
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Abstract
Three approaches were used to study hybridization of complementary oligodeoxynucleotides by nonradiative fluorescence resonance energy transfer. (i) Fluorescein (donor) and rhodamine (acceptor) were covalently attached to the 5' ends of complementary oligodeoxynucleotides of various lengths. Upon hybridization of the complementary oligodeoxynucleotides, energy transfer was detected by both a decrease in fluorescein emission intensity and an enhancement in rhodamine emission intensity. In all cases, fluorescein emission intensity was quenched by about 26% in the presence of unlabeled complement. Transfer efficiency at 5 degrees C decreased from 0.50 to 0.22 to 0.04 as the distance between donor and acceptor fluorophores in the hybrid increased from 8 to 12 to 16 nucleotides. Modeling of these hybrids as double helices showed that transfer efficiency decreased as the reciprocal of the sixth power of the donor-acceptor separation R, as predicted by theory with a corresponding R0 of 49 A. (ii) Fluorescence resonance energy transfer was used to study hybridization of two fluorophore-labeled oligonucleotides to a longer, unlabeled oligodeoxynucleotide. Two 12-mers were prepared that were complementary to two adjacent sequences separated by four bases on a 29-mer. The adjacent 5' and 3' ends of the two 12-mers labeled with fluorescein and rhodamine exhibited a transfer efficiency of approximately 0.60 at 5 degrees C when they both hybridized to the unlabeled 29-mer. (iii) An intercalating dye, acridine orange, was used as the donor fluorophore to a single rhodamine covalently attached to the 5' end of one oligodeoxynucleotide in a 12-base-pair hybrid. Under these conditions, the transfer efficiency was approximately 0.47 at 5 degrees C. These results establish that fluorescence modulation and nonradiative fluorescence resonance energy transfer can detect nucleic acid hybridization in solution. These techniques, with further development, may also prove useful for detecting and quantifying nucleic acid hybridization in living cells.
DOI: 10.1073/pnas.85.23.8790
PubMed: 3194390
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Detection of nucleic acid hybridization by nonradiative fluorescence resonance energy transfer.</title><author><name sortKey="Cardullo, R A" sort="Cardullo, R A" uniqKey="Cardullo R" first="R A" last="Cardullo">R A Cardullo</name><affiliation><nlm:affiliation>Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545.</nlm:affiliation></affiliation></author><author><name sortKey="Agrawal, S" sort="Agrawal, S" uniqKey="Agrawal S" first="S" last="Agrawal">S. Agrawal</name></author><author><name sortKey="Flores, C" sort="Flores, C" uniqKey="Flores C" first="C" last="Flores">C. Flores</name></author><author><name sortKey="Zamecnik, P C" sort="Zamecnik, P C" uniqKey="Zamecnik P" first="P C" last="Zamecnik">P C Zamecnik</name></author><author><name sortKey="Wolf, D E" sort="Wolf, D E" uniqKey="Wolf D" first="D E" last="Wolf">D E Wolf</name></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="1988">1988</date><idno type="RBID">pubmed:3194390</idno><idno type="pmid">3194390</idno><idno type="doi">10.1073/pnas.85.23.8790</idno><idno type="wicri:Area/PubMed/Corpus">002A50</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">002A50</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">Detection of nucleic acid hybridization by nonradiative fluorescence resonance energy transfer.</title><author><name sortKey="Cardullo, R A" sort="Cardullo, R A" uniqKey="Cardullo R" first="R A" last="Cardullo">R A Cardullo</name><affiliation><nlm:affiliation>Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545.</nlm:affiliation></affiliation></author><author><name sortKey="Agrawal, S" sort="Agrawal, S" uniqKey="Agrawal S" first="S" last="Agrawal">S. Agrawal</name></author><author><name sortKey="Flores, C" sort="Flores, C" uniqKey="Flores C" first="C" last="Flores">C. Flores</name></author><author><name sortKey="Zamecnik, P C" sort="Zamecnik, P C" uniqKey="Zamecnik P" first="P C" last="Zamecnik">P C Zamecnik</name></author><author><name sortKey="Wolf, D E" sort="Wolf, D E" uniqKey="Wolf D" first="D E" last="Wolf">D E Wolf</name></author></analytic><series><title level="j">Proceedings of the National Academy of Sciences of the United States of America</title><idno type="ISSN">0027-8424</idno><imprint><date when="1988" type="published">1988</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Chromatography, High Pressure Liquid</term><term>Energy Transfer</term><term>Fluorescent Dyes</term><term>Indicators and Reagents</term><term>Nucleic Acid Hybridization</term><term>Oligodeoxyribonucleotides</term><term>Spectrometry, Fluorescence (methods)</term></keywords><keywords scheme="MESH" type="chemical" xml:lang="en"><term>Fluorescent Dyes</term><term>Indicators and Reagents</term><term>Oligodeoxyribonucleotides</term></keywords><keywords scheme="MESH" qualifier="methods" xml:lang="en"><term>Spectrometry, Fluorescence</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Chromatography, High Pressure Liquid</term><term>Energy Transfer</term><term>Nucleic Acid Hybridization</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Three approaches were used to study hybridization of complementary oligodeoxynucleotides by nonradiative fluorescence resonance energy transfer. (i) Fluorescein (donor) and rhodamine (acceptor) were covalently attached to the 5' ends of complementary oligodeoxynucleotides of various lengths. Upon hybridization of the complementary oligodeoxynucleotides, energy transfer was detected by both a decrease in fluorescein emission intensity and an enhancement in rhodamine emission intensity. In all cases, fluorescein emission intensity was quenched by about 26% in the presence of unlabeled complement. Transfer efficiency at 5 degrees C decreased from 0.50 to 0.22 to 0.04 as the distance between donor and acceptor fluorophores in the hybrid increased from 8 to 12 to 16 nucleotides. Modeling of these hybrids as double helices showed that transfer efficiency decreased as the reciprocal of the sixth power of the donor-acceptor separation R, as predicted by theory with a corresponding R0 of 49 A. (ii) Fluorescence resonance energy transfer was used to study hybridization of two fluorophore-labeled oligonucleotides to a longer, unlabeled oligodeoxynucleotide. Two 12-mers were prepared that were complementary to two adjacent sequences separated by four bases on a 29-mer. The adjacent 5' and 3' ends of the two 12-mers labeled with fluorescein and rhodamine exhibited a transfer efficiency of approximately 0.60 at 5 degrees C when they both hybridized to the unlabeled 29-mer. (iii) An intercalating dye, acridine orange, was used as the donor fluorophore to a single rhodamine covalently attached to the 5' end of one oligodeoxynucleotide in a 12-base-pair hybrid. Under these conditions, the transfer efficiency was approximately 0.47 at 5 degrees C. These results establish that fluorescence modulation and nonradiative fluorescence resonance energy transfer can detect nucleic acid hybridization in solution. These techniques, with further development, may also prove useful for detecting and quantifying nucleic acid hybridization in living cells.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">3194390</PMID><DateCompleted><Year>1988</Year><Month>12</Month><Day>30</Day></DateCompleted><DateRevised><Year>2019</Year><Month>05</Month><Day>01</Day></DateRevised><Article PubModel="Print"><Journal><ISSN IssnType="Print">0027-8424</ISSN><JournalIssue CitedMedium="Print"><Volume>85</Volume><Issue>23</Issue><PubDate><Year>1988</Year><Month>Dec</Month></PubDate></JournalIssue><Title>Proceedings of the National Academy of Sciences of the United States of America</Title><ISOAbbreviation>Proc. Natl. Acad. Sci. U.S.A.</ISOAbbreviation></Journal><ArticleTitle>Detection of nucleic acid hybridization by nonradiative fluorescence resonance energy transfer.</ArticleTitle><Pagination><MedlinePgn>8790-4</MedlinePgn></Pagination><Abstract><AbstractText>Three approaches were used to study hybridization of complementary oligodeoxynucleotides by nonradiative fluorescence resonance energy transfer. (i) Fluorescein (donor) and rhodamine (acceptor) were covalently attached to the 5' ends of complementary oligodeoxynucleotides of various lengths. Upon hybridization of the complementary oligodeoxynucleotides, energy transfer was detected by both a decrease in fluorescein emission intensity and an enhancement in rhodamine emission intensity. In all cases, fluorescein emission intensity was quenched by about 26% in the presence of unlabeled complement. Transfer efficiency at 5 degrees C decreased from 0.50 to 0.22 to 0.04 as the distance between donor and acceptor fluorophores in the hybrid increased from 8 to 12 to 16 nucleotides. Modeling of these hybrids as double helices showed that transfer efficiency decreased as the reciprocal of the sixth power of the donor-acceptor separation R, as predicted by theory with a corresponding R0 of 49 A. (ii) Fluorescence resonance energy transfer was used to study hybridization of two fluorophore-labeled oligonucleotides to a longer, unlabeled oligodeoxynucleotide. Two 12-mers were prepared that were complementary to two adjacent sequences separated by four bases on a 29-mer. The adjacent 5' and 3' ends of the two 12-mers labeled with fluorescein and rhodamine exhibited a transfer efficiency of approximately 0.60 at 5 degrees C when they both hybridized to the unlabeled 29-mer. (iii) An intercalating dye, acridine orange, was used as the donor fluorophore to a single rhodamine covalently attached to the 5' end of one oligodeoxynucleotide in a 12-base-pair hybrid. Under these conditions, the transfer efficiency was approximately 0.47 at 5 degrees C. These results establish that fluorescence modulation and nonradiative fluorescence resonance energy transfer can detect nucleic acid hybridization in solution. These techniques, with further development, may also prove useful for detecting and quantifying nucleic acid hybridization in living cells.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Cardullo</LastName><ForeName>R A</ForeName><Initials>RA</Initials><AffiliationInfo><Affiliation>Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Agrawal</LastName><ForeName>S</ForeName><Initials>S</Initials></Author><Author ValidYN="Y"><LastName>Flores</LastName><ForeName>C</ForeName><Initials>C</Initials></Author><Author ValidYN="Y"><LastName>Zamecnik</LastName><ForeName>P C</ForeName><Initials>PC</Initials></Author><Author ValidYN="Y"><LastName>Wolf</LastName><ForeName>D E</ForeName><Initials>DE</Initials></Author></AuthorList><Language>eng</Language><GrantList CompleteYN="Y"><Grant><GrantID>HD07017</GrantID><Acronym>HD</Acronym><Agency>NICHD NIH HHS</Agency><Country>United States</Country></Grant><Grant><GrantID>HD07312</GrantID><Acronym>HD</Acronym><Agency>NICHD NIH HHS</Agency><Country>United States</Country></Grant><Grant><GrantID>P30-12708</GrantID><Agency>PHS HHS</Agency><Country>United States</Country></Grant></GrantList><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType><PublicationType UI="D013487">Research Support, U.S. Gov't, P.H.S.</PublicationType></PublicationTypeList></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>Proc Natl Acad Sci U S A</MedlineTA><NlmUniqueID>7505876</NlmUniqueID><ISSNLinking>0027-8424</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D005456">Fluorescent Dyes</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D007202">Indicators and Reagents</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D009838">Oligodeoxyribonucleotides</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D002851" MajorTopicYN="N">Chromatography, High Pressure Liquid</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D004735" MajorTopicYN="N">Energy Transfer</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D005456" MajorTopicYN="N">Fluorescent Dyes</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D007202" MajorTopicYN="N">Indicators and Reagents</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D009693" MajorTopicYN="Y">Nucleic Acid Hybridization</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D009838" MajorTopicYN="Y">Oligodeoxyribonucleotides</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D013050" MajorTopicYN="N">Spectrometry, Fluorescence</DescriptorName><QualifierName UI="Q000379" MajorTopicYN="N">methods</QualifierName></MeshHeading></MeshHeadingList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="pubmed"><Year>1988</Year><Month>12</Month><Day>1</Day></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>1988</Year><Month>12</Month><Day>1</Day><Hour>0</Hour><Minute>1</Minute></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>1988</Year><Month>12</Month><Day>1</Day><Hour>0</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">3194390</ArticleId><ArticleId IdType="pmc">PMC282592</ArticleId><ArticleId IdType="doi">10.1073/pnas.85.23.8790</ArticleId></ArticleIdList><ReferenceList><Reference><Citation>Nucleic Acids Res. 1986 Aug 11;14(15):6227-45</Citation><ArticleIdList><ArticleId IdType="pubmed">3748808</ArticleId></ArticleIdList></Reference><Reference><Citation>Proc Natl Acad Sci U S A. 1960 Jun;46(6):811-22</Citation><ArticleIdList><ArticleId IdType="pubmed">16590676</ArticleId></ArticleIdList></Reference><Reference><Citation>Anal Biochem. 1988 Feb 15;169(1):1-25</Citation><ArticleIdList><ArticleId IdType="pubmed">3285726</ArticleId></ArticleIdList></Reference><Reference><Citation>Biochemistry. 1983 Jul 5;22(14):3466-74</Citation><ArticleIdList><ArticleId IdType="pubmed">6225455</ArticleId></ArticleIdList></Reference><Reference><Citation>J Biomol Struct Dyn. 1987 Aug;5(1):127-43</Citation><ArticleIdList><ArticleId IdType="pubmed">3271462</ArticleId></ArticleIdList></Reference><Reference><Citation>Proc Natl Acad Sci U S A. 1967 Aug;58(2):719-26</Citation><ArticleIdList><ArticleId IdType="pubmed">5233469</ArticleId></ArticleIdList></Reference><Reference><Citation>J Cell Biol. 1986 Oct;103(4):1221-34</Citation><ArticleIdList><ArticleId IdType="pubmed">3771633</ArticleId></ArticleIdList></Reference></ReferenceList></PubmedData></pubmed></record>Pour manipuler ce document sous Unix (Dilib)
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