Spectroscopic characterization of fluorescein- and tetramethylrhodamine-labeled oligonucleotides and their complexes with a DNA template
We measured absorption and emission spectra, fluorescence quantum yield, anisotropy, fluorescence resonance energy transfer (FRET), and melting temperature to characterize fluorescein- and tetramethylrhodamine (TMR)-labeled oligonucleotides in solution and when hybridized to a common DNA template. U...
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Veröffentlicht in: | Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy Molecular and biomolecular spectroscopy, 2004-10, Vol.60 (12), p.2741-2750 |
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creator | Wang, L. Gaigalas, A.K. Blasic, J. Holden, M.J. |
description | We measured absorption and emission spectra, fluorescence quantum yield, anisotropy, fluorescence resonance energy transfer (FRET), and melting temperature to characterize fluorescein- and tetramethylrhodamine (TMR)-labeled oligonucleotides in solution and when hybridized to a common DNA template. Upon hybridization to the template, both the absorption and emission spectra of TMR-labeled duplexes exhibited a shift with respect to those of labeled oligonucleotides, depending on the location of the TMR on the oligonucleotide. Measurements of quantum yield, anisotropy, and melting temperature indicated that TMR interacted with nucleotides within the duplexes in the order (T1>T5>T11, T16) that the oligonucleotide with TMR labeled at the 5′ end (T1) is stronger than that labeled at position 5 from the 5′ end (T5), which is also stronger than those labeled at the positions, 11 and 16, from the 5′ end (T11, T16). In the case of the duplex formed between T1 and the template, fluorescence quenching was observed, which is attributed to the interaction between the dye molecule and guanosines located at the single-stranded portion of the template. A two-state model was suggested to describe the conformational states of TMR in the duplex. The melting temperatures of the four FRET complexes show the same pattern as those of TMR-labeled duplexes. We infer that the interactions between TMR and guanosine persist in the FRET complexes. This interaction may bring the donor and the acceptor molecules closely together, which could cause interaction between the two dye molecules shown in absorbance measurements of the FRET complexes. |
doi_str_mv | 10.1016/j.saa.2004.01.013 |
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Upon hybridization to the template, both the absorption and emission spectra of TMR-labeled duplexes exhibited a shift with respect to those of labeled oligonucleotides, depending on the location of the TMR on the oligonucleotide. Measurements of quantum yield, anisotropy, and melting temperature indicated that TMR interacted with nucleotides within the duplexes in the order (T1>T5>T11, T16) that the oligonucleotide with TMR labeled at the 5′ end (T1) is stronger than that labeled at position 5 from the 5′ end (T5), which is also stronger than those labeled at the positions, 11 and 16, from the 5′ end (T11, T16). In the case of the duplex formed between T1 and the template, fluorescence quenching was observed, which is attributed to the interaction between the dye molecule and guanosines located at the single-stranded portion of the template. A two-state model was suggested to describe the conformational states of TMR in the duplex. The melting temperatures of the four FRET complexes show the same pattern as those of TMR-labeled duplexes. We infer that the interactions between TMR and guanosine persist in the FRET complexes. This interaction may bring the donor and the acceptor molecules closely together, which could cause interaction between the two dye molecules shown in absorbance measurements of the FRET complexes.</description><identifier>ISSN: 1386-1425</identifier><identifier>DOI: 10.1016/j.saa.2004.01.013</identifier><identifier>PMID: 15350908</identifier><language>eng</language><publisher>England: Elsevier B.V</publisher><subject>Base Sequence ; DNA - chemistry ; DNA complexes ; Fluorescein ; Fluorescein - chemistry ; Fluorescence quenching ; Fluorescence resonance energy transfer ; Fluorescent Dyes ; Melting temperature ; Models, Chemical ; Molecular Structure ; Nucleic Acid Hybridization ; Oligodeoxyribonucleotides - chemistry ; Rhodamines - chemistry ; Spectrometry, Fluorescence ; Spectrophotometry ; Tetramethylrhodamine</subject><ispartof>Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy, 2004-10, Vol.60 (12), p.2741-2750</ispartof><rights>2004 Elsevier B.V.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c349t-9c3d01e8aff59ecf64411212f95d46f246d7cefe65d62bd6ef8dd5c5f94316253</citedby><cites>FETCH-LOGICAL-c349t-9c3d01e8aff59ecf64411212f95d46f246d7cefe65d62bd6ef8dd5c5f94316253</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.saa.2004.01.013$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3550,27924,27925,45995</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/15350908$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Wang, L.</creatorcontrib><creatorcontrib>Gaigalas, A.K.</creatorcontrib><creatorcontrib>Blasic, J.</creatorcontrib><creatorcontrib>Holden, M.J.</creatorcontrib><title>Spectroscopic characterization of fluorescein- and tetramethylrhodamine-labeled oligonucleotides and their complexes with a DNA template</title><title>Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy</title><addtitle>Spectrochim Acta A Mol Biomol Spectrosc</addtitle><description>We measured absorption and emission spectra, fluorescence quantum yield, anisotropy, fluorescence resonance energy transfer (FRET), and melting temperature to characterize fluorescein- and tetramethylrhodamine (TMR)-labeled oligonucleotides in solution and when hybridized to a common DNA template. Upon hybridization to the template, both the absorption and emission spectra of TMR-labeled duplexes exhibited a shift with respect to those of labeled oligonucleotides, depending on the location of the TMR on the oligonucleotide. Measurements of quantum yield, anisotropy, and melting temperature indicated that TMR interacted with nucleotides within the duplexes in the order (T1>T5>T11, T16) that the oligonucleotide with TMR labeled at the 5′ end (T1) is stronger than that labeled at position 5 from the 5′ end (T5), which is also stronger than those labeled at the positions, 11 and 16, from the 5′ end (T11, T16). In the case of the duplex formed between T1 and the template, fluorescence quenching was observed, which is attributed to the interaction between the dye molecule and guanosines located at the single-stranded portion of the template. A two-state model was suggested to describe the conformational states of TMR in the duplex. The melting temperatures of the four FRET complexes show the same pattern as those of TMR-labeled duplexes. We infer that the interactions between TMR and guanosine persist in the FRET complexes. This interaction may bring the donor and the acceptor molecules closely together, which could cause interaction between the two dye molecules shown in absorbance measurements of the FRET complexes.</description><subject>Base Sequence</subject><subject>DNA - chemistry</subject><subject>DNA complexes</subject><subject>Fluorescein</subject><subject>Fluorescein - chemistry</subject><subject>Fluorescence quenching</subject><subject>Fluorescence resonance energy transfer</subject><subject>Fluorescent Dyes</subject><subject>Melting temperature</subject><subject>Models, Chemical</subject><subject>Molecular Structure</subject><subject>Nucleic Acid Hybridization</subject><subject>Oligodeoxyribonucleotides - chemistry</subject><subject>Rhodamines - chemistry</subject><subject>Spectrometry, Fluorescence</subject><subject>Spectrophotometry</subject><subject>Tetramethylrhodamine</subject><issn>1386-1425</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2004</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kU9r3DAQxXVIyN9-gFyCTr15o7Et1SankKRNIbSHpGehlUa1FtlyJbnt5hPkY1dhF3oLPBgY3nswvyHkAtgKGIirzSoptaoZa1cMipoDcgJNJypoa35MTlPaMMagq9kROQbecNaz7oS8Ps2ocwxJh9lpqgcVlc4Y3YvKLkw0WGr9EiImjW6qqJoMzZijGjEPWx-HYNToJqy8WqNHQ4N3P8O0aI8hO4NplxjQRarDOHv8W3Z_XB6oonffbkpZWaqM5-TQKp_ww36ekR-f759vH6rH71--3t48Vrpp-1z1ujEMsFPW8h61FW0LUENte25aYetWmE8aLQpuRL02Am1nDNfc9m0DoubNGfm4651j-LVgynJ05Tbv1YRhSVKIjrMORDHCzqgLnRTRyjm6UcWtBCbfkMuNLMjlG3LJoKgpmct9-bIe0fxP7HkXw_XOgOXE3w6jTNrhpNG4WP4gTXDv1P8DYjiXqA</recordid><startdate>20041001</startdate><enddate>20041001</enddate><creator>Wang, L.</creator><creator>Gaigalas, A.K.</creator><creator>Blasic, J.</creator><creator>Holden, M.J.</creator><general>Elsevier B.V</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>20041001</creationdate><title>Spectroscopic characterization of fluorescein- and tetramethylrhodamine-labeled oligonucleotides and their complexes with a DNA template</title><author>Wang, L. ; Gaigalas, A.K. ; Blasic, J. ; Holden, M.J.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c349t-9c3d01e8aff59ecf64411212f95d46f246d7cefe65d62bd6ef8dd5c5f94316253</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2004</creationdate><topic>Base Sequence</topic><topic>DNA - chemistry</topic><topic>DNA complexes</topic><topic>Fluorescein</topic><topic>Fluorescein - chemistry</topic><topic>Fluorescence quenching</topic><topic>Fluorescence resonance energy transfer</topic><topic>Fluorescent Dyes</topic><topic>Melting temperature</topic><topic>Models, Chemical</topic><topic>Molecular Structure</topic><topic>Nucleic Acid Hybridization</topic><topic>Oligodeoxyribonucleotides - chemistry</topic><topic>Rhodamines - chemistry</topic><topic>Spectrometry, Fluorescence</topic><topic>Spectrophotometry</topic><topic>Tetramethylrhodamine</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Wang, L.</creatorcontrib><creatorcontrib>Gaigalas, A.K.</creatorcontrib><creatorcontrib>Blasic, J.</creatorcontrib><creatorcontrib>Holden, M.J.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Wang, L.</au><au>Gaigalas, A.K.</au><au>Blasic, J.</au><au>Holden, M.J.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Spectroscopic characterization of fluorescein- and tetramethylrhodamine-labeled oligonucleotides and their complexes with a DNA template</atitle><jtitle>Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy</jtitle><addtitle>Spectrochim Acta A Mol Biomol Spectrosc</addtitle><date>2004-10-01</date><risdate>2004</risdate><volume>60</volume><issue>12</issue><spage>2741</spage><epage>2750</epage><pages>2741-2750</pages><issn>1386-1425</issn><abstract>We measured absorption and emission spectra, fluorescence quantum yield, anisotropy, fluorescence resonance energy transfer (FRET), and melting temperature to characterize fluorescein- and tetramethylrhodamine (TMR)-labeled oligonucleotides in solution and when hybridized to a common DNA template. Upon hybridization to the template, both the absorption and emission spectra of TMR-labeled duplexes exhibited a shift with respect to those of labeled oligonucleotides, depending on the location of the TMR on the oligonucleotide. Measurements of quantum yield, anisotropy, and melting temperature indicated that TMR interacted with nucleotides within the duplexes in the order (T1>T5>T11, T16) that the oligonucleotide with TMR labeled at the 5′ end (T1) is stronger than that labeled at position 5 from the 5′ end (T5), which is also stronger than those labeled at the positions, 11 and 16, from the 5′ end (T11, T16). In the case of the duplex formed between T1 and the template, fluorescence quenching was observed, which is attributed to the interaction between the dye molecule and guanosines located at the single-stranded portion of the template. A two-state model was suggested to describe the conformational states of TMR in the duplex. The melting temperatures of the four FRET complexes show the same pattern as those of TMR-labeled duplexes. We infer that the interactions between TMR and guanosine persist in the FRET complexes. This interaction may bring the donor and the acceptor molecules closely together, which could cause interaction between the two dye molecules shown in absorbance measurements of the FRET complexes.</abstract><cop>England</cop><pub>Elsevier B.V</pub><pmid>15350908</pmid><doi>10.1016/j.saa.2004.01.013</doi><tpages>10</tpages></addata></record> |
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subjects | Base Sequence DNA - chemistry DNA complexes Fluorescein Fluorescein - chemistry Fluorescence quenching Fluorescence resonance energy transfer Fluorescent Dyes Melting temperature Models, Chemical Molecular Structure Nucleic Acid Hybridization Oligodeoxyribonucleotides - chemistry Rhodamines - chemistry Spectrometry, Fluorescence Spectrophotometry Tetramethylrhodamine |
title | Spectroscopic characterization of fluorescein- and tetramethylrhodamine-labeled oligonucleotides and their complexes with a DNA template |
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