SELEX Selection of High-Affinity Oligonucleotides for Bacteriophage Ff Gene 5 Protein†
Identifieur interne : 000D62 ( Istex/Corpus ); précédent : 000D61; suivant : 000D63SELEX Selection of High-Affinity Oligonucleotides for Bacteriophage Ff Gene 5 Protein†
Auteurs : Jin-Der Wen ; Carla W. Gray ; Donald M. GraySource :
- Biochemistry [ 0006-2960 ] ; 2001.
Abstract
The Ff gene 5 protein (g5p) is a cooperative ssDNA-binding protein. SELEX was used to identify DNA sequences favorable for g5p binding at physiological ionic strength (200 mM NaCl) and 37 °C. Sequences were selected from a library of 58-mers that contained a central variable segment of 26 nucleotides. DNA sequences selected after eight rounds of SELEX were mostly G-rich, with multiple copies of CPuGGPy, TPuGGGPy, and/or PyPuPuGGGPy motifs. This was unexpected, since g5p has higher binding affinities for polypyrimidine than for polypurine sequences. The most recurrent G-rich sequence, named I-3, was found to have g5p-binding properties that were correlated with a structural transition. At 10 mM NaCl, I-3 existed in a single-stranded form that was saturated by g5p in an all-or-none fashion. At 200 mM NaCl, I-3 existed in a structured form that showed CD spectral features of G-quadruplexes. The g5p binding affinity for this structured form of I-3 was >100-fold higher than for the single-stranded form. Moreover, the structured I-3 was saturated by g5p in two steps, the first of which was the formation of an apparent initiation complex consisting of one I-3 strand and about three g5p dimers. Nuclease S1 footprinting and other experiments showed that g5p molecules in the initiation complex at 200 mM NaCl were bound directly to the G-rich variable segment and that the structure of I-3 was retained after saturation by g5p. Thus, G-rich motifs may form structures favorable for initiation of g5p binding and also provide the actual g5p-binding sites.
Url:
DOI: 10.1021/bi010109z
Links to Exploration step
ISTEX:373A64E897640F4570DDCACBFFC388C995F2720CLe document en format XML
<record><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title>SELEX Selection of High-Affinity Oligonucleotides for Bacteriophage Ff Gene 5 Protein†</title><author><name sortKey="Wen, Jin Der" sort="Wen, Jin Der" uniqKey="Wen J" first="Jin-Der" last="Wen">Jin-Der Wen</name><affiliation><mods:affiliation>Department of Molecular and Cell Biology, The University of Texas at Dallas, Box 830688, Richardson, Texas 75083-0688</mods:affiliation></affiliation></author><author><name sortKey="Gray, Carla W" sort="Gray, Carla W" uniqKey="Gray C" first="Carla W." last="Gray">Carla W. Gray</name><affiliation><mods:affiliation>Department of Molecular and Cell Biology, The University of Texas at Dallas, Box 830688, Richardson, Texas 75083-0688</mods:affiliation></affiliation></author><author><name sortKey="Gray, Donald M" sort="Gray, Donald M" uniqKey="Gray D" first="Donald M." last="Gray">Donald M. Gray</name><affiliation><mods:affiliation>Department of Molecular and Cell Biology, The University of Texas at Dallas, Box 830688, Richardson, Texas 75083-0688</mods:affiliation></affiliation><affiliation><mods:affiliation> To whom correspondence should be addressed. Department ofMolecular and Cell Biology, Mail Stop FO 3.1, The University of Texasat Dallas, Box 830688, Richardson, TX 75083-0688; (972) 883-2513;FAX (972) 883-2409; e-mail: dongray@utdallas.edu.</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:373A64E897640F4570DDCACBFFC388C995F2720C</idno><date when="2001" year="2001">2001</date><idno type="doi">10.1021/bi010109z</idno><idno type="url">https://api.istex.fr/ark:/67375/TPS-KD58MTD9-V/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">000D62</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">000D62</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main">SELEX Selection of High-Affinity Oligonucleotides for Bacteriophage Ff Gene 5
Protein<ref type="bib" target="#bi010109zAF2"><hi rend="superscript">†</hi></ref></title><author><name sortKey="Wen, Jin Der" sort="Wen, Jin Der" uniqKey="Wen J" first="Jin-Der" last="Wen">Jin-Der Wen</name><affiliation><mods:affiliation>Department of Molecular and Cell Biology, The University of Texas at Dallas, Box 830688, Richardson, Texas 75083-0688</mods:affiliation></affiliation></author><author><name sortKey="Gray, Carla W" sort="Gray, Carla W" uniqKey="Gray C" first="Carla W." last="Gray">Carla W. Gray</name><affiliation><mods:affiliation>Department of Molecular and Cell Biology, The University of Texas at Dallas, Box 830688, Richardson, Texas 75083-0688</mods:affiliation></affiliation></author><author><name sortKey="Gray, Donald M" sort="Gray, Donald M" uniqKey="Gray D" first="Donald M." last="Gray">Donald M. Gray</name><affiliation><mods:affiliation>Department of Molecular and Cell Biology, The University of Texas at Dallas, Box 830688, Richardson, Texas 75083-0688</mods:affiliation></affiliation><affiliation><mods:affiliation> To whom correspondence should be addressed. Department ofMolecular and Cell Biology, Mail Stop FO 3.1, The University of Texasat Dallas, Box 830688, Richardson, TX 75083-0688; (972) 883-2513;FAX (972) 883-2409; e-mail: dongray@utdallas.edu.</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j" type="main">Biochemistry</title><title level="j" type="abbrev">Biochemistry</title><idno type="ISSN">0006-2960</idno><idno type="eISSN">1520-4995</idno><imprint><publisher>American Chemical Society</publisher><date type="e-published">2001</date><date type="published">2001</date><biblScope unit="vol">40</biblScope><biblScope unit="issue">31</biblScope><biblScope unit="page" from="9300">9300</biblScope><biblScope unit="page" to="9310">9310</biblScope></imprint><idno type="ISSN">0006-2960</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0006-2960</idno></seriesStmt></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract">The Ff gene 5 protein (g5p) is a cooperative ssDNA-binding protein. SELEX was used to identify DNA sequences favorable for g5p binding at physiological ionic strength (200 mM NaCl) and 37 °C. Sequences were selected from a library of 58-mers that contained a central variable segment of 26 nucleotides. DNA sequences selected after eight rounds of SELEX were mostly G-rich, with multiple copies of CPuGGPy, TPuGGGPy, and/or PyPuPuGGGPy motifs. This was unexpected, since g5p has higher binding affinities for polypyrimidine than for polypurine sequences. The most recurrent G-rich sequence, named I-3, was found to have g5p-binding properties that were correlated with a structural transition. At 10 mM NaCl, I-3 existed in a single-stranded form that was saturated by g5p in an all-or-none fashion. At 200 mM NaCl, I-3 existed in a structured form that showed CD spectral features of G-quadruplexes. The g5p binding affinity for this structured form of I-3 was >100-fold higher than for the single-stranded form. Moreover, the structured I-3 was saturated by g5p in two steps, the first of which was the formation of an apparent initiation complex consisting of one I-3 strand and about three g5p dimers. Nuclease S1 footprinting and other experiments showed that g5p molecules in the initiation complex at 200 mM NaCl were bound directly to the G-rich variable segment and that the structure of I-3 was retained after saturation by g5p. 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Department ofMolecular and Cell Biology, Mail Stop FO 3.1, The University of Texasat Dallas, Box 830688, Richardson, TX 75083-0688; (972) 883-2513;FAX (972) 883-2409; e-mail: dongray@utdallas.edu.</json:string></affiliations></json:item></author><arkIstex>ark:/67375/TPS-KD58MTD9-V</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>research-article</json:string></originalGenre><abstract>The Ff gene 5 protein (g5p) is a cooperative ssDNA-binding protein. SELEX was used to identify DNA sequences favorable for g5p binding at physiological ionic strength (200 mM NaCl) and 37 °C. Sequences were selected from a library of 58-mers that contained a central variable segment of 26 nucleotides. DNA sequences selected after eight rounds of SELEX were mostly G-rich, with multiple copies of CPuGGPy, TPuGGGPy, and/or PyPuPuGGGPy motifs. This was unexpected, since g5p has higher binding affinities for polypyrimidine than for polypurine sequences. The most recurrent G-rich sequence, named I-3, was found to have g5p-binding properties that were correlated with a structural transition. At 10 mM NaCl, I-3 existed in a single-stranded form that was saturated by g5p in an all-or-none fashion. At 200 mM NaCl, I-3 existed in a structured form that showed CD spectral features of G-quadruplexes. The g5p binding affinity for this structured form of I-3 was >100-fold higher than for the single-stranded form. Moreover, the structured I-3 was saturated by g5p in two steps, the first of which was the formation of an apparent initiation complex consisting of one I-3 strand and about three g5p dimers. Nuclease S1 footprinting and other experiments showed that g5p molecules in the initiation complex at 200 mM NaCl were bound directly to the G-rich variable segment and that the structure of I-3 was retained after saturation by g5p. Thus, G-rich motifs may form structures favorable for initiation of g5p binding and also provide the actual g5p-binding sites.</abstract><qualityIndicators><score>9.976</score><pdfWordCount>8924</pdfWordCount><pdfCharCount>49862</pdfCharCount><pdfVersion>1.2</pdfVersion><pdfPageCount>11</pdfPageCount><pdfPageSize>612 x 792 pts (letter)</pdfPageSize><pdfWordsPerPage>811</pdfWordsPerPage><pdfText>true</pdfText><refBibsNative>true</refBibsNative><abstractWordCount>248</abstractWordCount><abstractCharCount>1565</abstractCharCount><keywordCount>0</keywordCount></qualityIndicators><title>SELEX Selection of High-Affinity Oligonucleotides for Bacteriophage Ff Gene 5 Protein†</title><genre><json:string>research-article</json:string></genre><host><title>Biochemistry</title><language><json:string>unknown</json:string></language><issn><json:string>0006-2960</json:string></issn><eissn><json:string>1520-4995</json:string></eissn><volume>40</volume><issue>31</issue><pages><first>9300</first><last>9310</last></pages><genre><json:string>journal</json:string></genre></host><ark><json:string>ark:/67375/TPS-KD58MTD9-V</json:string></ark><categories><wos><json:string>1 - science</json:string><json:string>2 - biochemistry & molecular biology</json:string></wos><scienceMetrix><json:string>1 - health sciences</json:string><json:string>2 - biomedical research</json:string><json:string>3 - biochemistry & molecular biology</json:string></scienceMetrix><scopus><json:string>1 - Life Sciences</json:string><json:string>2 - Biochemistry, Genetics and Molecular Biology</json:string><json:string>3 - Biochemistry</json:string></scopus><inist><json:string>1 - sciences appliquees, technologies et medecines</json:string><json:string>2 - sciences biologiques et medicales</json:string><json:string>3 - sciences biologiques fondamentales et appliquees. psychologie</json:string></inist></categories><publicationDate>2001</publicationDate><copyrightDate>2001</copyrightDate><doi><json:string>10.1021/bi010109z</json:string></doi><id>373A64E897640F4570DDCACBFFC388C995F2720C</id><score>1</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-KD58MTD9-V/fulltext.pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-KD58MTD9-V/bundle.zip</uri></json:item><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-KD58MTD9-V/fulltext.txt</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/TPS-KD58MTD9-V/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main">SELEX Selection of High-Affinity Oligonucleotides for Bacteriophage Ff Gene 5
Protein<ref type="bib" target="#bi010109zAF2"><hi rend="superscript">†</hi></ref></title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher>American Chemical Society</publisher><availability><licence>Copyright © 2001 American Chemical Society</licence><p>American Chemical Society</p></availability><date type="published">2001</date><date type="Copyright" when="2001">2001</date></publicationStmt><notesStmt><note type="content-type" source="research-article" scheme="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</note><note type="publication-type" scheme="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</note></notesStmt><sourceDesc><biblStruct type="article"><analytic><title level="a" type="main">SELEX Selection of High-Affinity Oligonucleotides for Bacteriophage Ff Gene 5
Protein<ref type="bib" target="#bi010109zAF2"><hi rend="superscript">†</hi></ref></title><author xml:id="author-0000"><persName><surname>Wen</surname><forename type="first">Jin-Der</forename></persName><affiliation><orgName type="laboratory">Department of Molecular and Cell Biology</orgName><orgName type="institution">The University of Texas at Dallas</orgName><address><addrLine>Box 830688</addrLine><addrLine>Richardson</addrLine><addrLine>Texas 75083-0688</addrLine></address></affiliation></author><author xml:id="author-0001"><persName><surname>Gray</surname><forename type="first">Carla W.</forename></persName><affiliation><orgName type="laboratory">Department of Molecular and Cell Biology</orgName><orgName type="institution">The University of Texas at Dallas</orgName><address><addrLine>Box 830688</addrLine><addrLine>Richardson</addrLine><addrLine>Texas 75083-0688</addrLine></address></affiliation></author><author xml:id="author-0002" role="corresp"><persName><surname>Gray</surname><forename type="first">Donald M.</forename></persName><affiliation><orgName type="laboratory">Department of Molecular and Cell Biology</orgName><orgName type="institution">The University of Texas at Dallas</orgName><address><addrLine>Box 830688</addrLine><addrLine>Richardson</addrLine><addrLine>Texas 75083-0688</addrLine></address></affiliation><affiliation role="corresp"> To whom correspondence should be addressed. Department of Molecular and Cell Biology, Mail Stop FO 3.1, The University of Texas at Dallas, Box 830688, Richardson, TX 75083-0688; (972) 883-2513; FAX (972) 883-2409; e-mail: dongray@utdallas.edu.</affiliation></author><idno type="istex">373A64E897640F4570DDCACBFFC388C995F2720C</idno><idno type="ark">ark:/67375/TPS-KD58MTD9-V</idno><idno type="DOI">10.1021/bi010109z</idno></analytic><monogr><title level="j" type="main">Biochemistry</title><title level="j" type="abbrev">Biochemistry</title><idno type="acspubs">bi</idno><idno type="coden">bichaw</idno><idno type="pISSN">0006-2960</idno><idno type="eISSN">1520-4995</idno><imprint><publisher>American Chemical Society</publisher><date type="e-published">2001</date><date type="published">2001</date><biblScope unit="vol">40</biblScope><biblScope unit="issue">31</biblScope><biblScope unit="page" from="9300">9300</biblScope><biblScope unit="page" to="9310">9310</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><encodingDesc><schemaRef type="ODD" url="https://xml-schema.delivery.istex.fr/tei-istex.odd"></schemaRef><appInfo><application ident="pub2tei" version="1.0.41" when="2020-04-06"><label>pub2TEI-ISTEX</label><desc>A set of style sheets for converting XML documents encoded in various scientific publisher formats into a common TEI format.
<ref target="http://www.tei-c.org/">We use TEI</ref></desc></application></appInfo></encodingDesc><profileDesc><abstract><p>The Ff gene 5 protein (g5p) is a cooperative ssDNA-binding protein. SELEX was used to
identify DNA sequences favorable for g5p binding at physiological ionic strength (200 mM NaCl) and
37 °C. Sequences were selected from a library of 58-mers that contained a central variable segment of 26
nucleotides. DNA sequences selected after eight rounds of SELEX were mostly G-rich, with multiple
copies of CPuGGPy, TPuGGGPy, and/or PyPuPuGGGPy motifs. This was unexpected, since g5p has
higher binding affinities for polypyrimidine than for polypurine sequences. The most recurrent G-rich
sequence, named I-3, was found to have g5p-binding properties that were correlated with a structural
transition. At 10 mM NaCl, I-3 existed in a single-stranded form that was saturated by g5p in an all-or-none fashion. At 200 mM NaCl, I-3 existed in a structured form that showed CD spectral features of
G-quadruplexes. The g5p binding affinity for this structured form of I-3 was >100-fold higher than for
the single-stranded form. Moreover, the structured I-3 was saturated by g5p in two steps, the first of
which was the formation of an apparent initiation complex consisting of one I-3 strand and about three
g5p dimers. Nuclease S1 footprinting and other experiments showed that g5p molecules in the initiation
complex at 200 mM NaCl were bound directly to the G-rich variable segment and that the structure of I-3
was retained after saturation by g5p. Thus, G-rich motifs may form structures favorable for initiation of
g5p binding and also provide the actual g5p-binding sites.
</p></abstract><textClass ana="subject"><keywords scheme="document-type-name"><term>Article</term></keywords></textClass><langUsage><language ident="en"></language></langUsage></profileDesc><revisionDesc><change when="2020-04-06" who="#istex" xml:id="pub2tei">formatting</change></revisionDesc></teiHeader></istex:fulltextTEI></fulltext><metadata><istex:metadataXml wicri:clean="corpus acs not found" wicri:toSee="no header"><istex:xmlDeclaration>version="1.0" encoding="UTF-8"</istex:xmlDeclaration><istex:document><article article-type="research-article" specific-use="acs2jats-1.1.23" dtd-version="1.1d1"><front><journal-meta><journal-id journal-id-type="acspubs">bi</journal-id><journal-id journal-id-type="coden">bichaw</journal-id><journal-title-group><journal-title>Biochemistry</journal-title><abbrev-journal-title>Biochemistry</abbrev-journal-title></journal-title-group><issn pub-type="ppub">0006-2960</issn><issn pub-type="epub">1520-4995</issn><publisher><publisher-name>American Chemical Society</publisher-name></publisher><self-uri>pubs.acs.org/biochemistry</self-uri></journal-meta><article-meta><article-id pub-id-type="doi">10.1021/bi010109z</article-id><article-categories><subj-group subj-group-type="document-type-name"><subject>Article</subject></subj-group></article-categories><title-group><article-title>SELEX Selection of High-Affinity Oligonucleotides for Bacteriophage Ff Gene 5
Protein<xref rid="bi010109zAF2"><sup>†</sup></xref></article-title></title-group><contrib-group><contrib contrib-type="author"><name name-style="western"><surname>Wen</surname><given-names>Jin-Der</given-names></name></contrib><contrib contrib-type="author"><name name-style="western"><surname>Gray</surname><given-names>Carla W.</given-names></name></contrib><contrib contrib-type="author" corresp="yes"><name name-style="western"><surname>Gray</surname><given-names>Donald M.</given-names></name><xref rid="bi010109zAF1">*</xref></contrib><aff>Department of Molecular and Cell Biology, The University of Texas at Dallas, Box 830688, Richardson, Texas 75083-0688
</aff></contrib-group><author-notes><corresp id="bi010109zAF1">
To whom correspondence should be addressed. Department of
Molecular and Cell Biology, Mail Stop FO 3.1, The University of Texas
at Dallas, Box 830688, Richardson, TX 75083-0688; (972) 883-2513;
FAX (972) 883-2409; e-mail: dongray@utdallas.edu.</corresp></author-notes><pub-date pub-type="epub"><day>12</day><month>07</month><year>2001</year></pub-date><pub-date pub-type="ppub"><day>07</day><month>08</month><year>2001</year></pub-date><volume>40</volume><issue>31</issue><fpage>9300</fpage><lpage>9310</lpage><history><date date-type="received"><day>17</day><month>01</month><year>2001</year></date><date date-type="rev-recd"><day>03</day><month>05</month><year>2001</year></date><date date-type="asap"><day>12</day><month>07</month><year>2001</year></date><date date-type="issue-pub"><day>07</day><month>08</month><year>2001</year></date></history><permissions><copyright-statement>Copyright © 2001 American Chemical Society</copyright-statement><copyright-year>2001</copyright-year><copyright-holder>American Chemical Society</copyright-holder></permissions><abstract><p>The Ff gene 5 protein (g5p) is a cooperative ssDNA-binding protein. SELEX was used to
identify DNA sequences favorable for g5p binding at physiological ionic strength (200 mM NaCl) and
37 °C. Sequences were selected from a library of 58-mers that contained a central variable segment of 26
nucleotides. DNA sequences selected after eight rounds of SELEX were mostly G-rich, with multiple
copies of CPuGGPy, TPuGGGPy, and/or PyPuPuGGGPy motifs. This was unexpected, since g5p has
higher binding affinities for polypyrimidine than for polypurine sequences. The most recurrent G-rich
sequence, named I-3, was found to have g5p-binding properties that were correlated with a structural
transition. At 10 mM NaCl, I-3 existed in a single-stranded form that was saturated by g5p in an all-or-none fashion. At 200 mM NaCl, I-3 existed in a structured form that showed CD spectral features of
G-quadruplexes. The g5p binding affinity for this structured form of I-3 was >100-fold higher than for
the single-stranded form. Moreover, the structured I-3 was saturated by g5p in two steps, the first of
which was the formation of an apparent initiation complex consisting of one I-3 strand and about three
g5p dimers. Nuclease S1 footprinting and other experiments showed that g5p molecules in the initiation
complex at 200 mM NaCl were bound directly to the G-rich variable segment and that the structure of I-3
was retained after saturation by g5p. Thus, G-rich motifs may form structures favorable for initiation of
g5p binding and also provide the actual g5p-binding sites.
</p></abstract><custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name><meta-value>bi010109z</meta-value></custom-meta></custom-meta-group></article-meta><notes id="bi010109zAF2"><label>†</label><p>
This work was performed by J.-D.W. in partial fulfillment of the
requirements for the Ph.D. degree in the Department of Molecular and
Cell Biology, The University of Texas at Dallas. Support was provided
by grants from the Robert A. Welch Foundation (AT-503) and the Texas
Advanced Technology Program (009741-0021-1999).</p></notes></front><body><sec id="d7e123"><title></title><p>The genome of the Ff phages<xref rid="bi010109zb00001" ref-type="bibr"></xref> comprises 11 tightly packed
genes and an intergenic region between genes 4 and 2 (<italic toggle="yes"><xref rid="bi010109zb00001" ref-type="bibr"></xref></italic>).
The Ff g5p is a single-stranded DNA binding protein of
monomer MW 9690 (87 amino acid residues) that exists as
a stable dimer even at a concentration as low as 1 nM (<italic toggle="yes"><xref rid="bi010109zb00002" ref-type="bibr"></xref></italic>).
The g5p dimer has 2-fold rotational symmetry and cooperatively saturates the Ff ssDNA genome by binding antiparallel-stranded nucleotides in its dyadic DNA-binding sites
(<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi010109zb00003" ref-type="bibr"></xref>−<xref rid="bi010109zb00004" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi010109zb00005" ref-type="bibr"></xref></named-content></italic>). There are three modes of binding, in which 4, 3, or
2.5 nucleotides are bound per g5p monomer (i.e., <italic toggle="yes">n</italic> = 4, 3,
or 2.5; <italic toggle="yes">6</italic>,<italic toggle="yes"> 7</italic>). The <italic toggle="yes">n</italic> = 4 mode of binding is the dominant
mode when g5p is present at P/N ratios ≤ 0.25. However,
there is virtually no information available on how g5p
initiates cooperative binding to the viral genome under
physiological conditions. The ssDNA complexed with g5p
does not serve as a template for dsDNA synthesis and is a
precursor for virion assembly (<italic toggle="yes"><xref rid="bi010109zb00001" ref-type="bibr"></xref></italic>).
</p><p>Owing to its biological functions in saturating the viral
genome, g5p has been most studied for its non-sequence-specific binding properties (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi010109zb00003" ref-type="bibr"></xref>, <xref rid="bi010109zb00008" ref-type="bibr"></xref></named-content></italic>). However, g5p is known
to have marked differences in binding affinities (Kω) for
synthetic single-stranded polynucleotides (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi010109zb00009" ref-type="bibr"></xref>, <xref rid="bi010109zb00010" ref-type="bibr"></xref></named-content></italic>). A polynucleotide that is more stacked, like the polypurine poly[d(A)], binds with lower affinity than do the less stacked
polypyrimidines poly[d(T)] and poly[d(C)]. Moreover, g5p
has structure- and sequence-specific binding functions of
biological importance. (a) The complexes isolated from cells
frequently have three or four branches, suggestive of local
preferential initiation at more than one site (C. W. Gray,
unpublished results). (b) A (G+C)-rich hairpin of 32 base
pairs in the intergenic region is oriented at one end of the
g5p·ssDNA intracellular complex (<italic toggle="yes"><xref rid="bi010109zb00011" ref-type="bibr"></xref></italic>) and maintains the
same orientation in the mature virus after being packaged
(<italic toggle="yes"><xref rid="bi010109zb00012" ref-type="bibr"></xref></italic>). (c) The g5p inhibits the “−” strand synthesis and
synthesis of replicative form (RF) dsDNA, but not merely
by sequestering genomic “+” strands. Fulford and Model
(<italic toggle="yes"><xref rid="bi010109zb00013" ref-type="bibr"></xref></italic>) proposed a competitive melting by g5p and stabilization
by g2p of hairpins at the “−” strand origin as a switching
mechanism that controls synthesis of RF dsDNA. (d) Fulford
and Model (<italic toggle="yes"><xref rid="bi010109zb00013" ref-type="bibr"></xref></italic>) also showed that low levels of g5p provide
immunity to Ff superinfection, inconsistent with simple
saturation of the infecting ssDNA by g5p. (e) The g5p binds
to the mRNA leader sequences of genes 1, 2, 3, 5, and 10 to
regulate their translation (<italic toggle="yes"><xref rid="bi010109zb00014" ref-type="bibr"></xref></italic>). Binding sites in the gene 2
and 3 leader sequences contain G-rich blocks of four or five
purines surrounded by blocks of pyrimidines. (f) The g5p
has been shown to repress the translation of gene 2 mRNA
both in vitro and in vivo (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi010109zb00015" ref-type="bibr"></xref>, <xref rid="bi010109zb00016" ref-type="bibr"></xref></named-content></italic>). Michel and Zinder (<italic toggle="yes"><xref rid="bi010109zb00017" ref-type="bibr"></xref></italic>)
have defined a sequence of 16 nucleotides in the gene 2
mRNA that is required in vivo for repression by g5p. They
also found that, in vitro, an RNA carrying this sequence is
at least 10-fold higher in affinity for g5p binding than is an
RNA lacking it. The preferential binding of g5p to an RNA
carrying the 16-mer sequence is affected by mutations that
abolish gene 2 translational repression in vivo (<italic toggle="yes"><xref rid="bi010109zb00018" ref-type="bibr"></xref></italic>). (g) A
direct measurement by mass spectrometry shows that two
g5p dimers bind to a DNA analogue of this 16-mer RNA,
bending it to form a hairpin (<italic toggle="yes"><xref rid="bi010109zb00019" ref-type="bibr"></xref></italic>). Therefore, as shown by
these examples, g5p plays a role in regulation of viral DNA
synthesis and viral gene expression, probably through
structure- and/or sequence-specific binding.
</p><p>The SELEX methodology was originally developed to
select from an in vitro library (a pool of RNA sequences
with a randomized region and two flanking constant sequences) those sequences having high affinity for a protein
(<italic toggle="yes"><xref rid="bi010109zb00020" ref-type="bibr"></xref></italic>) or immobilized dyes (<italic toggle="yes"><xref rid="bi010109zb00021" ref-type="bibr"></xref></italic>). SELEX has also been applied
to the selection of DNA molecules from double- and single-stranded DNA libraries. The targets that have been used for
SELEX include (a) nucleic acid-binding proteins, (b) proteins
that are not thought to bind nucleic acids naturally, and (c)
small molecules such as nucleotides, amino acids, cofactors,
and dyes (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi010109zb00022" ref-type="bibr"></xref>−<xref rid="bi010109zb00023" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi010109zb00024" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi010109zb00025" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi010109zb00026" ref-type="bibr"></xref></named-content></italic>). The first example of SELEX using an
ssDNA library was with thrombin, a protease that was
considered not to interact physiologically with nucleic acids
(<italic toggle="yes"><xref rid="bi010109zb00027" ref-type="bibr"></xref></italic>). Aptamers of ssDNA that bind thrombin with high
affinities display a highly conserved region of 14−17
nucleotides, of which one typical sequence folds into a
unimolecular quadruplex containing two G-quartets, as
determined by NMR spectroscopy (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi010109zb00028" ref-type="bibr"></xref>, <xref rid="bi010109zb00029" ref-type="bibr"></xref></named-content></italic>).
</p><p>In this report, we present the first application of SELEX
using a cooperative ssDNA binding protein, the Ff g5p. The
g5p binds with a large, positive cooperativity factor, ω, so
that the protein tends to saturate all nucleic acid sequences.
Nevertheless, our results show that the SELEX strategy can
be used to efficiently select high-affinity ssDNA sequences.
The gist of our results is that the initiation of cooperative
binding, when the binding is least dependent on ω, can be
very dependent on the ssDNA sequence and structure.
</p></sec><sec id="d7e231"><title>Experimental Procedures</title><p><italic toggle="yes">SELEX, PCR, and Sequencing.</italic> The SELEX procedure
applied in this paper was derived from that used by Gold
and co-workers (<italic toggle="yes"><xref rid="bi010109zb00030" ref-type="bibr"></xref></italic>). A synthesized library of 58-mer ssDNA,
called PV-58, was used for SELEX selection. PV-58 DNA
was synthesized with the following sequence: 5‘-CGGGATCCAACGTTTT-N<sub>26</sub>-AAGAGGCAGAATTCGC-3‘ (Oligos Etc.). A, G, C, and T were randomly incorporated in
the central 26 nucleotides (N<sub>26</sub>). The two flanking constant
16-mer sequences were used as primer annealing sites for
PCR amplification. The g5p was isolated and purified as in
previous work (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi010109zb00007" ref-type="bibr"></xref>, <xref rid="bi010109zb00010" ref-type="bibr"></xref></named-content></italic>).
</p><p>For the initial selection, 1 nmol (6 × 10<sup>14</sup> sequences) of
PV-58 (a small portion was <sup>32</sup>P-labeled at the 5‘ end with
T4 polynucleotide kinase; see below) was incubated with
g5p in 200 mM NaCl at 37 °C for 15 min in TE buffer (10
mM Tris-HCl, pH 7.4, 1 mM EDTA). The amount of g5p
was adjusted to give a selection ratio (complexed DNA/total
DNA) of 0.005 to 0.05 for each round of selection. G5p·DNA complexes were separated from free DNAs on the basis
of EMSA (see below), except that the gels were not fixed.
Instead, the resulting wet gel was exposed briefly to a storage
phosphor screen (Molecular Dynamics). Superimposing the
gel with the printed image enabled the position of complexed
DNA to be located. The band corresponding to a saturated
complex was excised from the wet agarose gel and the
excised gel slice was liquefied by treatment with β-agarase
(FMC BioProducts) at 45 °C for 1−2 h. The g5p-bound
DNA was extracted by phenol/chloroform and then ethanol-precipitated.
</p><p>PCR was performed to amplify the selected DNA with 5‘
primer (5‘-CGGGATCCAACGTTTT-3‘) and biotin-conjugated 3‘ primer (biotin-5‘-GCGAATTCTGCCTCTT-3‘) (Midland) under the following conditions: 95 °C for 2 min; 16
cycles at 95 °C for 1 min, 50 °C for 1 min, and 72 °C for 2
min; the final extension was at 72 °C for 10 min. [α<sup>32</sup>P]dCTP (ICN) was included in the PCR mixture to internally
label the DNA. PCR products were purified in agarose gels
with the <italic toggle="yes">QIAEX II</italic> kit (QIAGEN). To isolate the target
ssDNA (extended with the 5‘ primer), an immobilized
streptavidin agarose bead matrix (Pierce) was added to bind
the purified biotin-conjugated dsDNA, which was then
denatured by 0.12 N NaOH at 37 °C for 15 min. Since the
biotin−streptavidin interaction was not disrupted by this
alkaline solution, only the target ssDNA was released from
the matrix (<italic toggle="yes"><xref rid="bi010109zb00030" ref-type="bibr"></xref></italic>). The ssDNA was finally recovered by ethanol
precipitation. Generally, 200−300 pmol of the enriched
ssDNA was used for the following round of SELEX.
</p><p>DNAs from the fourth, sixth, and eighth rounds of
selection, as well as the original sample of PV-58, were
cloned with the <italic toggle="yes">TOPO TA Cloning</italic> kit (Invitrogen) and
sequenced with the <italic toggle="yes">fmol DNA Sequencing System</italic> (Promega).
</p><p><italic toggle="yes">EMSA.</italic> The I-3 DNA sequence (5‘-CGGGATCCAACGTTTT-GGGGTCAGGCTGGGGTTGTGCAGGTC-AAGAGGCAGAATTCGC-3‘) (Oligos Etc.) was <sup>32</sup>P-labeled at the 5‘ end
using T4 polynucleotide kinase (Invitrogen) and [γ<sup>32</sup>P]ATP
(ICN). For titrations with g5p, 1 μM of labeled I-3 was
incubated with 0−21 μM of g5p at 37 °C for 15 min in TE
buffer containing 10 or 200 mM NaCl. Mixtures were loaded
on 2.5% low-melting agarose gels (FMC BioProducts) in
TAE buffer (40 mM Tris-acetate, pH 8.3, 1 mM EDTA),
followed by electrophoresis at 8−10 V/cm for 80 min. DNA
that was complexed with g5p had reduced electrophoretic
mobility and was shifted to higher molecular-weight positions
on gels. The gels were fixed with 10% acetic acid/50%
methanol for 2 h, dried, and exposed to a storage phosphor
screen. The bands were quantitated and analyzed with
ImageQuant, v. 5.0 (Molecular Dynamics).
</p><p>For competition experiments, 1 μM of <sup>32</sup>P-labeled I-3 was
incubated in TE buffer with 14 μM of g5p in the absence or
presence of 2 μM of an unlabeled competitor, I-7, at 37 °C
for 15 min. I-7 (Oligos Etc.) differed from I-3 in that its
central 26 nucleotide segment had the sequence 5‘-GTGCCACCCTCCTCTCTTGTTCTTGT-3‘. The NaCl concentrations were 10, 50, 100, and 200 mM. After electrophoresis
of the samples, the gels were fixed and quantitated as
described above.
</p><p>To determine the apparent binding affinities, Kω<sub>app</sub>, for a
g5p dimer, EMSA titrations of ssDNA (I-3 or PV-58) with
g5p were quantitated and the apparent g5p binding affinity
was determined as Kω<sub>app</sub> = 1/(2<italic toggle="yes">L</italic>), where <italic toggle="yes">L</italic> is the free g5p
dimer concentration at which 50% of labeled DNA was
saturated with g5p. This binding affinity is for binding of a
g5p dimer in one orientation, and the binding is assumed to
be all-or-none (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi010109zb00007" ref-type="bibr"></xref>, <xref rid="bi010109zb00010" ref-type="bibr"></xref></named-content></italic>). Free g5p concentrations were estimated using the assumption that four nucleotides were bound
per g5p monomer in the <italic toggle="yes">n</italic> = 4 binding mode.
</p><p><italic toggle="yes">CD Measurements.</italic> CD spectra were measured in a Jasco
model J710 spectropolarimeter, smoothed, and plotted at
1-nm intervals as molar CD (ε<sub>L</sub> − ε<sub>R</sub>) in units of M<sup>-1</sup> cm<sup>-1</sup>,
per mole of nucleotide, as in previous work (<italic toggle="yes"><xref rid="bi010109zb00010" ref-type="bibr"></xref></italic>), except that
spectra were smoothed by the method of fast Fourier
transformation (Jasco).
</p><p><italic toggle="yes">Primer-Annealing and Serial Dilution Experiments.</italic> To
determine the strand stoichiometry of I-3 in its free state
and in the initiation complex with g5p, 1 μM of <sup>32</sup>P-labeled
I-3 was incubated with 0−4 μM of the 3‘ primer in 200 mM
NaCl at 37 °C for 30 min. For the formation of complexes,
g5p was added to give a final concentration of 7 μM for the
final 15 min of the incubation. Control experiments were
performed by reverse additions, preincubating I-3 with g5p
for 15 min prior to the addition of the 3‘ primer for another
15-min incubation. The 3‘ primer sequence was the one
described above but without conjugated biotin. Mixtures were
subjected to electrophoresis (10 V/cm) in 12% polyacrylamide (acrylamide/bis = 19:1) gels in TBE buffer (90 mM
Tris-borate, pH 8.3, 2 mM EDTA) for 4 h. Gels were fixed
with 10% acetic acid for 10 min, dried, and exposed to a
storage phosphor screen.
</p><p>For serial dilutions, I-3 was diluted in 10 or 200 mM NaCl
to final concentrations of 4, 1, 0.25, 0.063, and 0.016 μM
strand. A trace amount of <sup>32</sup>P-labeled I-3 was added to each
dilution. Samples were heated at 90 °C for 3 min, cooled at
room temperature, and then subjected to electrophoresis as
described above.
</p><p><italic toggle="yes">Stoichiometry of Protein and DNA in g5p•I-3 Complexes.</italic>
To determine the stoichiometric ratio of protein to DNA,
g5p and I-3 in the complexes were quantitated together by
the following procedure: <sup>32</sup>P-labeled I-3 was mixed with g5p
at 10 mM NaCl (6 μM I-3 per 32 μM g5p) or 200 mM NaCl
(12.5 μM I-3 per 80 μM g5p). Mixtures were incubated at
37 °C for 15 min and electrophoresed in low-melting agarose
gels as described above. The bands of g5p·I-3 complexes
were isolated and heated with SDS loading buffer at final
concentrations of 50 mM Tris-HCl, pH 6.8, 100 mM
dithiothreitol, 2% SDS, 0.1% bromophenol blue, and 10%
glycerol. The melted agarose solution was loaded on a 15%
polyacrylamide gel, and SDS−PAGE was performed by the
method of Laemmli (<italic toggle="yes"><xref rid="bi010109zb00031" ref-type="bibr"></xref></italic>). A series of standard amounts of
g5p and <sup>32</sup>P-labeled I-3 were also run in the same gel. For
g5p quantitation, the gel was stained with SYPRO Red
(BioWhittaker Molecular Applications; <italic toggle="yes">32</italic>) for 2 h, destained
with 7.5% acetic acid for 10 min, and scanned with a
STORM 860 (red fluorescence mode; Molecular Dynamics).
For I-3 quantitation, the same gel was dried and exposed to
a storage phosphor screen. Calibration curves were plotted
with the standards, and the respective amounts of g5p and
I-3 in the complexes were calculated.
</p><p><italic toggle="yes">Quantitative Nuclease S1 Footprinting.</italic> To determine the
initiation sites on structured I-3 (in 200 mM NaCl), nuclease
S1 (Promega) digestion was carried out under conditions such
that only the initiation “band 2” complex existed and the
saturated “band 1” complex did not. 1 μM of 5‘ end <sup>32</sup>P-labeled I-3 was incubated in the presence or absence of 1.4
μM g5p in 10 μL of 200 mM NaCl (in 10 mM Tris-HCl,
pH 7.4) at 37 °C for 15 min. A nuclease S1 mixture was
added to give a final concentration of 0.67 unit/μL of
nuclease S1 and 1 mM ZnCl<sub>2</sub>. The reaction mixture was
incubated for 1 min and stopped by adding an equal volume
of 88% formamide and 30 mM EDTA. The nuclease-digested
samples were briefly heated and then resolved in 12%
denaturing polyacrylamide gels (containing 7.6 M urea;
acrylamide/bis = 19:1). After being dried, the gel was
exposed to a storage phosphor screen. The bands were
quantitated, and their positions were assigned according to
four parallel reference sequencing lanes of A, G, C, and T.
</p><p>Since the amount of I-3 complexed with g5p under these
conditions accounted for only 10% of the total I-3 (see
Results), most signals were contributed by free I-3, and
quantitative analysis was needed. Each band of the g5p-bound I-3 and the control lane was quantitated, and the
percent protection of the corresponding base by g5p was
calculated according to the following equation: 100% ×
(quantity in band without g5p − quantity in band with g5p)/(quantity in band without g5p). Those sequence positions
protected by g5p gave positive values of the percent
protection.
</p><p><italic toggle="yes">Determination of End Boundaries of I-3 in the Initiation
Complex.</italic> To determine the 3‘-end boundary of the region
of I-3 needed to form the initiation complex with g5p, I-3
was <sup>32</sup>P-labeled at the 5‘ end with T4 polynucleotide kinase.
Partial nuclease S1 (0.1 unit/μL) digestion was performed
at room temperature for 5 min to generate random I-3
fragments. I-3 fragments at a total nucleotide concentration
≈ 50 μM were incubated with 0−14 μM g5p at 37 °C for
15 min in TE buffer containing 200 mM NaCl. The mixtures
were applied to a membrane filter (0.45 μm, HAWP,
containing nitrocellulose; Millipore) to selectively bind
protein and protein-containing complexes. The bound DNA
was extracted from the filter with phenol/chloroform, ethanol-precipitated, and then resolved in 12% denaturing polyacrylamide gels. The gels were dried and exposed to a storage
phosphor screen. Band positions were assigned according
to four parallel reference sequencing lanes of A, G, C, and
T.
</p><p>For 5‘-end boundary determination, I-3 was labeled at the
3‘ end by employing T4 RNA ligase (Promega) and [5‘-<sup>32</sup>P]pCp (ICN). The same procedure described above was applied
except that the range of g5p concentrations was 0−40 μM.
Since specific length markers were not available, band
positions were assigned by counting each band of the
fragment starting from the intact I-3 on a high-resolution
phosphor image. This assignment was based on the assumption that each phosphodiester bond of I-3 was accessible to
nuclease S1.
</p></sec><sec id="d7e391"><title>Results</title><p><italic toggle="yes">ssDNA Sequences Selected using SELEX.</italic> SELEX was
used to select high-affinity g5p-binding sequences from an
ssDNA library of 58-mers, PV-58, that contained a central
stretch of 26 nucleotides with approximately random incorporation of the four nucleotides. One nanomole of PV-58 (6
× 10<sup>14</sup> sequences) was used for the initial selection. To
approximate physiological conditions, the selection was
performed at 37 °C in a buffer containing 200 mM NaCl,
pH 7.4. After eight rounds, 36 sequences were cloned and
sequenced (Table <xref rid="bi010109zt00001"></xref>). Nineteen clones had an identical
sequence (named “I-3”), and seven additional ones differed
from I-3 by one base. Although g5p is known to prefer to
bind to pyrimidines, 32 of the 36 independently cloned
oligonucleotides surprisingly were G-rich. G-centered motifs
with three to five purines, CPuGGPy, TPuGGGPy, and
PyPuPuGGGPy (Pu stands for A or G, and Py for C or T),
repeatedly appeared among most of these sequences (Table
<xref rid="bi010109zt00001"></xref>). The information content (<italic toggle="yes"><xref rid="bi010109zb00033" ref-type="bibr"></xref></italic>) for the variable region after
selection was 25.3 bits, which was an average of 0.97 bit
per position. Since the information content of the unselected
material averaged only 0.07 bit per position, there was a
significant enrichment of sequences during the selection
procedure. For example, if each position comprised 80% of
a specific base and 20% of equal numbers of the other three
bases, the information content would be 0.96 bit per position.
For the calculation of information content, all the sequences
were aligned as shown in Table <xref rid="bi010109zt00001"></xref>, were weighted by the
number of identical clones, and were corrected for sampling
uncertainty (<italic toggle="yes"><xref rid="bi010109zb00033" ref-type="bibr"></xref></italic>).
<table-wrap id="bi010109zt00001" position="float" orientation="portrait"><label>1</label><caption><p>36 ssDNA Sequences Recovered after Eight Rounds of SELEX Selection</p></caption><oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="1"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry colname="1"><graphic xlink:href="bi010109zu00001a.tif" position="float" orientation="portrait"></graphic></oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic><sup></sup> Clones for sequencing were designated by a letter for the agar plate and a number for the clone from that plate. Where more than one clone
had the same sequence, the sequence is named for the first clone.<italic toggle="yes"><sup>b</sup></italic><sup></sup> Only the sequences of the N<sub>26</sub> variable region are shown from 5‘ to 3‘. Numbering
is from the 5‘ end of the complete PV-58 sequence. The “t” at the 5‘ end of some sequences is from the constant region. Repeated motifs: boxed
for CPuGGPy, shaded for TPuGGGPy, and double-underlined for PyPuPuGGGPy (Pu stands for A or G; Py for C or T).<italic toggle="yes"><sup>c</sup></italic><sup></sup> Bases in just the variable
region.<italic toggle="yes"><sup>d</sup></italic><sup></sup> The figures in parentheses refer to the numbers of clones with identical sequences.</p></table-wrap-foot></table-wrap></p><p>The predominant sequence I-3 had two TG<sub>4</sub>T and two
CAG<sub>2</sub>Py motifs, which gave I-3 an unexpected resemblance
to a combination of the <italic toggle="yes">Tetrahymena</italic> telomeric repeat (TG<sub>4</sub>T;
<italic toggle="yes">34</italic>) and the human telomeric repeat (TAG<sub>3</sub>T; <italic toggle="yes">35</italic>). I-3 was
chemically synthesized for further characterization.
</p><p>To explore the evolution of the G-rich sequences during
SELEX, analyses of the cloned sequences and their base
distribution for the intermediate rounds of selection were
performed. As shown in Table <xref rid="bi010109zt00002"></xref>, the variable region of the
synthetic PV-58 library was slightly higher in G content than
the expected 25%. A preference for guanine in randomly
synthesized DNA has been reported elsewhere (<italic toggle="yes"><xref rid="bi010109zb00024" ref-type="bibr"></xref></italic>). After
four rounds of selection, each of the 24 cloned sequences
was pyrimidine-rich and the G content dropped to 13%. Thus,
the early rounds of selection were for moderately high
affinity pyrimidine-rich sequences, as expected. Then, the
guanine population increased dramatically between the sixth
and eighth rounds. The final selected sequences had specific
G-rich patterns that evolved from the SELEX procedure and
were different from the majority of G-rich sequences in the
PV-58 library.
<table-wrap id="bi010109zt00002" position="float" orientation="portrait"><label>2</label><caption><p>Base Distributions in the Variable N<sub>26</sub> Region after
Different Numbers of Rounds of SELEX</p></caption><oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="8"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:colspec colnum="2" colname="2"></oasis:colspec><oasis:colspec colnum="3" colname="3"></oasis:colspec><oasis:colspec colnum="4" colname="4"></oasis:colspec><oasis:colspec colnum="5" colname="5"></oasis:colspec><oasis:colspec colnum="6" colname="6"></oasis:colspec><oasis:colspec colnum="7" colname="7"></oasis:colspec><oasis:colspec colnum="8" colname="8"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2"></oasis:entry><oasis:entry namest="3" nameend="8">averaged base population
per strand (%)</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">SELEX
round</oasis:entry><oasis:entry colname="2"># of cloned
sequences</oasis:entry><oasis:entry colname="3">A</oasis:entry><oasis:entry colname="4">G</oasis:entry><oasis:entry colname="5">C</oasis:entry><oasis:entry colname="6">T</oasis:entry><oasis:entry colname="7">Pu</oasis:entry><oasis:entry colname="8">Py
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">0
</oasis:entry><oasis:entry colname="2">16
</oasis:entry><oasis:entry colname="3">18.5
</oasis:entry><oasis:entry colname="4">32.9
</oasis:entry><oasis:entry colname="5">22.4
</oasis:entry><oasis:entry colname="6">26.2
</oasis:entry><oasis:entry colname="7">51.4
</oasis:entry><oasis:entry colname="8">48.6
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">4
</oasis:entry><oasis:entry colname="2">24
</oasis:entry><oasis:entry colname="3">16.2
</oasis:entry><oasis:entry colname="4">13.1
</oasis:entry><oasis:entry colname="5">35.9
</oasis:entry><oasis:entry colname="6">34.8
</oasis:entry><oasis:entry colname="7">29.3
</oasis:entry><oasis:entry colname="8">70.7
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">6
</oasis:entry><oasis:entry colname="2">13
</oasis:entry><oasis:entry colname="3">11.2
</oasis:entry><oasis:entry colname="4">16.0
</oasis:entry><oasis:entry colname="5">41.7
</oasis:entry><oasis:entry colname="6">31.1
</oasis:entry><oasis:entry colname="7">27.2
</oasis:entry><oasis:entry colname="8">72.8
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">8
</oasis:entry><oasis:entry colname="2">36
</oasis:entry><oasis:entry colname="3">9.2
</oasis:entry><oasis:entry colname="4">49.8
</oasis:entry><oasis:entry colname="5">19.2
</oasis:entry><oasis:entry colname="6">21.8
</oasis:entry><oasis:entry colname="7">59.0
</oasis:entry><oasis:entry colname="8">41.0</oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table></table-wrap></p><p><italic toggle="yes">G5p Binds Differently to I-3 at 10 and 200 mM NaCl.</italic> In
initial experiments to determine the binding affinity of g5p
for I-3, I-3 titrations with g5p were analyzed by EMSA. The
I-3 (1 μM) was <sup>32</sup>P-labeled at the 5‘ end and incubated with
increasing concentrations (0 to 21 μM) of g5p in 10 and
200 mM NaCl at 37 °C (Figure <xref rid="bi010109zf00001"></xref>). At 10 mM NaCl, one
band of complex appeared (Figure <xref rid="bi010109zf00001"></xref>A). Since only a minor
amount of radioactivity (≤10%) appeared between the
complexed and free DNA bands, this binding was approximately all-or-none, reflecting the high cooperativity of
g5p. Because one band of complexed I-3 persisted throughout
the titration, it was identified as a saturated complex. A slight
retardation in the mobility of the saturated complex was
always observed in the presence of excess g5p, as shown in
lane 10 of Figure <xref rid="bi010109zf00001"></xref>A. This possibly was due to a well-known
switch in binding mode from <italic toggle="yes">n</italic> = 4 to <italic toggle="yes">n</italic> = 3 in the presence
of excess g5p (<italic toggle="yes"><xref rid="bi010109zb00006" ref-type="bibr"></xref></italic>).
<fig id="bi010109zf00001" position="float" orientation="portrait"><label>1</label><caption><p>EMSA of g5p titrations of I-3 in 10 and 200 mM NaCl.<sup>32</sup>P-labeled I-3 (1 μM) was titrated at 37 °C with increasing
concentrations of g5p in (A) 10 mM NaCl or (B) 200 mM NaCl.
Samples were subjected to 2.5% agarose gel electrophoresis in TAE
buffer. The gels were fixed, dried, and analyzed as described in
Experimental Procedures. The concentrations of g5p were (from
left to right in both panels) 0, 1.4, 2.8, 4.2, 5.6, 8.4, 11.2, 14, 16.8,
and 21 μM (per monomer). 14 μM of g5p (lane 8) was a
concentration theoretically sufficient to saturate I-3 in the <italic toggle="yes">n</italic> = 4
binding mode. That is, the [protein monomer]/[nucleotide] ratio
was P/N = 0.25 in lane 8.</p></caption><graphic xlink:href="bi010109zf00001.tif" position="float" orientation="portrait"></graphic></fig></p><p>At 200 mM NaCl, the salt concentration at which SELEX
selection was performed, we were surprised to observe an
additional band (band 2) of complex that was formed at low
g5p concentrations (Figure <xref rid="bi010109zf00001"></xref>B). The band 2 complex
appeared prior to the appearance of the saturated (band 1)
complex (Figure <xref rid="bi010109zf00001"></xref>B, lane 2), reached a plateau in the middle
of the titration (lanes 4−6), and essentially disappeared
before the end of titration (lane 8). The concentration of g5p
in lane 8 was sufficient to saturate the I-3 in an <italic toggle="yes">n</italic> = 4 binding
mode. Therefore, the band 2 complex appeared to be an
intermediate to the formation of a saturated band 1 complex.
Since the intermediate complex was not saturated with g5p,
we designate it an initiation complex.
</p><p><italic toggle="yes">Salt Effects on g5p-Binding Affinity of I-3.</italic> That the
selected DNA sequence I-3 could form different complexes
with g5p at different salt concentrations was unusual. The
binding affinity of g5p for I-3 was >100-fold higher at 200
mM NaCl than at 10 mM NaCl, whereas the affinity of g5p
for PV-58 was slightly reduced at the higher salt concentration (Table <xref rid="bi010109zt00003"></xref>). These results, combined with the results in
Figure <xref rid="bi010109zf00001"></xref>, suggested that the high affinity of g5p for I-3 was
achieved through the formation of the initiation complex at
200 mM NaCl.
<table-wrap id="bi010109zt00003" position="float" orientation="portrait"><label>3</label><caption><p>Apparent Binding Affinities of the g5p Dimer for I-3 and PV-58 in 10 and 200 mM NaCl</p></caption><oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="5"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:colspec colnum="2" colname="2"></oasis:colspec><oasis:colspec colnum="3" colname="3"></oasis:colspec><oasis:colspec colnum="4" colname="4"></oasis:colspec><oasis:colspec colnum="5" colname="5"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry namest="2" nameend="3">I-3</oasis:entry><oasis:entry namest="4" nameend="5">PV-58</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">NaCl</oasis:entry><oasis:entry colname="2">10 mM</oasis:entry><oasis:entry colname="3">200 mM</oasis:entry><oasis:entry colname="4">10 mM</oasis:entry><oasis:entry colname="5">200 mM
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">Kω<sub>app </sub> (× 10<sup>-5</sup> M<sup>-1</sup>)
</oasis:entry><oasis:entry colname="2">3.0 ± 0.7<italic toggle="yes"><sup>a</sup></italic><sup></sup></oasis:entry><oasis:entry colname="3">>300<italic toggle="yes"><sup>b</sup></italic><sup></sup></oasis:entry><oasis:entry colname="4">2.4 ± 0.2<italic toggle="yes"><sup>a</sup></italic><sup></sup></oasis:entry><oasis:entry colname="5">2.1 ± 0.1<italic toggle="yes"><sup>a</sup></italic><sup></sup></oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic><sup></sup> Values were obtained from EMSA titrations; see Experimental
Procedures. Data are the averages of two independent experiments.
Errors are the range of values from the two experiments.<italic toggle="yes"><sup>b</sup></italic><sup></sup> This is a
minimal value and was estimated from the concentration of PV-58
needed to dissociate 50% of the g5p•I-3 band 1 complex in a
competition experiment in 200 mM NaCl.</p></table-wrap-foot></table-wrap></p><p>EMSA was used to further explore the salt effects on the
binding affinity of g5p for I-3. The <sup>32</sup>P-labeled I-3 was
incubated with g5p and with increasing concentrations of
NaCl in the presence or absence of an unlabeled competitor,
I-7 (Figure <xref rid="bi010109zf00002"></xref>). I-7 was the most pyrimidine-rich sequence
obtained from the eighth round of SELEX; Table <xref rid="bi010109zt00001"></xref>. As
shown in Figure <xref rid="bi010109zf00002"></xref>, the competitiveness of I-7 for g5p binding
decreased with increasing concentrations of NaCl. Moreover,
the initiation complex (band 2) only appeared when the NaCl
concentration was ≥ 50 mM. Therefore, the g5p-binding
affinity of I-3 was salt-dependent, and the formation of the
initiation complex was correlated with higher affinity binding, relative to the pyrimidine-rich sequence I-7, at higher
salt concentrations.
<fig id="bi010109zf00002" position="float" orientation="portrait"><label>2</label><caption><p>Effects of NaCl concentration on g5p affinity for I-3. (A) EMSA of<sup>32</sup>P-labeled I-3 (1 μM) that was incubated with 14
μM of g5p at 37 °C for 15 min in the presence (lanes 2, 4, 6, and
8) or absence (lanes 1, 3, 5, and 7) of a competitor (2 μM of I-7).
The NaCl concentrations during the incubation were 10, 50, 100,
and 200 mM, as shown at the bottom of the figure. Gels were run
and analyzed as for Figure <xref rid="bi010109zf00001"></xref>. The band 2 complex appeared under
competitive conditions in which NaCl concentrations were at least
50 mM (lanes 4, 6, and 8). (B) Chart showing the relative
percentages of band 1 and band 2 complexes in lanes 2, 4, 6, and
8. The percentages were normalized to lanes 1, 3, 5, and 7,
respectively. The data were the averages of two independent
experiments, except that only one experiment was performed at
100 mM NaCl. The error bars show the range of values from the
two experiments.</p></caption><graphic xlink:href="bi010109zf00002.tif" position="float" orientation="portrait"></graphic></fig></p><p><italic toggle="yes">CD Titrations of I-3 with g5p at 10 and 200 mM NaCl.</italic>
CD titrations were used to further characterize the g5p-binding properties of I-3 at 10 and 200 mM NaCl at 37 °C.
As shown in Figure <xref rid="bi010109zf00003"></xref>A, the free I-3 (solid line) in 10 mM
NaCl showed a typical CD spectrum of single stranded DNA,
and the spectral changes above 245 nm upon addition of
g5p were monotonic and were similar to those found for
titrations of other ssDNAs with g5p at low salt concentrations
(<italic toggle="yes"><xref rid="bi010109zb00036" ref-type="bibr"></xref></italic>). The end point of this titration was at a P/N ratio of
0.25−0.34 (one g5p monomer per 4 to 3 nucleotides; Figure
<xref rid="bi010109zf00003"></xref>A, inset).
<fig id="bi010109zf00003" position="float" orientation="portrait"><label>3</label><caption><p>CD spectra of the full-length and truncated I-3. The full-length I-3 (about 1 μM strand concentration) was titrated with g5p in 10 mM NaCl (A) or 200 mM NaCl (B) at 37 °C. Representative spectra taken during the titrations are shown. (A) CD spectra at 10 mM NaCl of free I-3 () and g5p·I-3 complexes
at P/N ratios of 0.07 (○), 0.15 (▪), 0.25 (▵), and 0.34 (▾). (B) CD
spectra at 200 mM NaCl of free I-3 () and g5p·I-3 complexes at
P/N ratios of 0.02 (○), 0.06 (▪), 0.14 (▵), and 0.29 (▾). Insets
show CD values at 260 nm as a function of P/N ratio. (C) CD
spectra of the truncated I-3 in 10 mM KCl (○), 200 mM KCl (▪),
10 mM NaCl (▵), and 200 mM NaCl (▾) at 37 °C. All spectra are
plotted as ε<sub>L</sub> − ε<sub>R</sub> in units of M<sup>-1</sup> cm<sup>-1</sup>, per mole of nucleotide,
with values on the left-hand scales. In addition, the right-hand scale
for panel C shows nucleotide molar values reduced by a factor of
26/58, which allows a more direct comparison of band magnitudes
for the truncated I-3 sequence (panel C) and the full I-3 sequence
(panel B) when both are at 200 mM NaCl.</p></caption><graphic xlink:href="bi010109zf00003.tif" position="float" orientation="portrait"></graphic></fig></p><p>At 200 mM NaCl, CD titrations showed two modes of
binding, one at P/N ratios < 0.1 (less than one g5p monomer
per 10 nucleotides), followed by the stoichiometric saturation
of I-3 at a P/N ratio close to 0.25 (one g5p monomer per 4
nucleotides; Figure <xref rid="bi010109zf00003"></xref>B, inset). These binding modes were
consistent with the results of EMSA (see Figure <xref rid="bi010109zf00001"></xref>) and with
the formation of the initiation complex at low g5p concentrations (smaller P/N ratios), followed by the formation of the
saturated complex with increasing concentrations of g5p
(larger P/N ratios). In addition, the spectrum of free I-3 in
200 mM NaCl showed two positive bands at about 260 and
290 nm (see Figure <xref rid="bi010109zf00003"></xref>B, solid line). Since there are four copies
of telomeric and telomere-like sequence motifs in the variable
region of I-3 (Table <xref rid="bi010109zt00001"></xref>), I-3 may form an intrastranded
G-quartet structure (interstranded interaction was ruled out
because I-3 existed in a unimolecular form; see below).
Parallel G-quadruplexes have a characteristic CD spectrum
with a positive band at 260−265 nm (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi010109zb00037" ref-type="bibr"></xref>, <xref rid="bi010109zb00038" ref-type="bibr"></xref></named-content></italic>), while
antiparallel G-quadruplexes show a positive band at 290−295 nm and a negative band close to 260 nm (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi010109zb00037" ref-type="bibr"></xref>, <xref rid="bi010109zb00038" ref-type="bibr"></xref></named-content></italic>).
Therefore, unbound I-3 in 200 mM NaCl showed a combination of the CD spectral features of two types of G-quadruplexes. The overall spectrum of free I-3 was most like
that previously reported for an <italic toggle="yes">Oxytricha </italic>(T<sub>4</sub>G<sub>4</sub>)<sub>2</sub> hairpin (<italic toggle="yes"><xref rid="bi010109zb00039" ref-type="bibr"></xref></italic>).
</p><p>These two positive CD bands were present, but with
different intensities, when I-3 was saturated with g5p (see
Figure <xref rid="bi010109zf00003"></xref>B, P/N = 0.29), suggesting that the overall structure
of I-3 was not substantially altered by g5p binding.
</p><p><italic toggle="yes">CD Spectra of a Truncated I-3 Sequence.</italic> To further test
whether the G-rich central segment could be responsible for
the CD spectral features of I-3 in 200 mM NaCl, CD spectra
were obtained for a truncated I-3 sequence (5‘-GGGGTCAGGCTGGGGTTGTGCAGGTC-3‘), denoted I-3c26. This
sequence consisted of only the central 26 nucleotides of I-3
and contained 14 G's (see Table <xref rid="bi010109zt00001"></xref>). CD spectra of I-3c26
displayed large changes as the NaCl concentration was
increased from 10 to 200 mM. At 200 mM NaCl, I-3c26
acquired CD bands at about 255 and 292 nm (Figure <xref rid="bi010109zf00003"></xref>C,
solid triangles) that were close to those of the full-length
I-3 under the same conditions (Figure <xref rid="bi010109zf00003"></xref>B, solid line). The
magnitudes of the positive CD bands of I-3 and I-3c26 were
also in reasonable agreement (ranging from 0.9 to 1.4 M<sup>-1</sup>
cm<sup>-1</sup>), if the reduced number of nucleotides in the truncated
I-3c26 sequence was taken into account by multiplying the
I-3c26 spectrum by a factor of 26/58. (Compare the left-hand scale of Figure <xref rid="bi010109zf00003"></xref>B with the right-hand scale of Figure
<xref rid="bi010109zf00003"></xref>C). These CD data supported the view that the CD spectrum
of the I-3 sequence in 200 mM NaCl was dominated by
G-quartets within the central sequence of 26 nucleotides.
</p><p>The I-3c26 sequence can potentially form a variety of
structures containing G-quartets, depending on which G's
are involved. It is also known that G-quadruplex structures
are influenced by whether the cation is Na<sup>+</sup> or K<sup>+</sup> (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi010109zb00040" ref-type="bibr"></xref>, <xref rid="bi010109zb00041" ref-type="bibr"></xref></named-content></italic>).
Intramolecular chair-type structures appear to require the
presence of potassium (<italic toggle="yes"><xref rid="bi010109zb00041" ref-type="bibr"></xref></italic>). As I-3c26 was titrated from 10
to 200 mM KCl, the 292-nm positive band increased, a
negative band appeared at 260 nm, and a small positive band
appeared above 240 nm (Figure <xref rid="bi010109zf00003"></xref>C). These features were
remarkably similar to those in the CD spectrum of a thrombin
binding aptamer in 25 mM KCl (<italic toggle="yes"><xref rid="bi010109zb00042" ref-type="bibr"></xref></italic>), which is known to
fold into an intramolecular chair-form G-quadruplex in which
adjacent guanosines along and between strands alternate in
their glycosyl (<italic toggle="yes">syn</italic> or <italic toggle="yes">anti</italic>) conformations (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi010109zb00028" ref-type="bibr"></xref>, <xref rid="bi010109zb00029" ref-type="bibr"></xref></named-content></italic>). This
provided strong evidence that I-3c26 can indeed form a
G-quadruplex structure that is probably an intramolecular
chair fold. The specific G-quadruplex fold that might be
formed by I-3c26 and the full-length I-3 in the presence of
Na<sup>+</sup> ion is not known, but it could conceivably be an
intramolecular basket-type fold. The positive 260 nm CD
band in the presence of Na<sup>+</sup> could then have its origin in
the nonalternating arrangement of glycosyl bonds within the
G-tetramers in such a fold (<italic toggle="yes"><xref rid="bi010109zb00028" ref-type="bibr"></xref></italic>) and/or in a sodium-dependent
stacking of nontetrameric G's.
</p><p><italic toggle="yes">Stoichiometry of the Initiation Complex.</italic> Primer-annealing
experiments were performed to determine the strand stoichiometry of I-3 in the initiation complex. The method was
similar to that used by others to study G-quartet complexes
(<italic toggle="yes"><xref rid="bi010109zb00043" ref-type="bibr"></xref></italic>). <sup>32</sup>P-labeled I-3 was incubated with various concentrations of the 16-mer primer that was complementary to the
3‘ end of I-3. If I-3 existed as a unimolecular form, one
retarded band with I-3 plus an annealed primer, in addition
to the primer-free band, should appear during gel electrophoresis. If I-3 existed in an n-stranded form, up to n bands,
in addition to the primer-free band, could appear during
electrophoresis. Figure <xref rid="bi010109zf00004"></xref>A, lanes 1−4, shows that only one
additional band was detected when the 3‘ primer was
annealed to free I-3.
<fig id="bi010109zf00004" position="float" orientation="portrait"><label>4</label><caption><p>Stoichiometry of free and g5p-complexed I-3. (A) Primer annealing of free and g5p-complexed I-3. The 3‘ primer (0, 0.5, 2, and 4 μM, as shown at the bottom of the figure) was incubated with 1 μM of<sup>32</sup>P-labeled free I-3 (lanes 1−4) or I-3 complexed
with 7 μM g5p (lanes 5−8) in 200 mM NaCl buffer. Reaction
mixtures were resolved in 12% polyacrylamide gels in TBE buffer.
The gels were then fixed, dried, and analyzed. Details are given in
Experimental Procedures. (B) Serial dilution of free I-3. Four-fold
dilutions of I-3 were made in 200 mM NaCl (lanes 1−5) and 10
mM NaCl (lanes 6−10). A trace amount of <sup>32</sup>P-labeled I-3 was
added to each dilution. The final I-3 concentrations are shown at
the bottom of the figure. Electrophoresis was performed as for panel
A.</p></caption><graphic xlink:href="bi010109zf00004.tif" position="float" orientation="portrait"></graphic></fig></p><p>Figure <xref rid="bi010109zf00004"></xref>A, lanes 5−8, further shows that, after the addition
of g5p to form an initiation band 2 complex, only one additional band 2 complex appeared upon addition of the 3‘
primer. This strongly suggested that free I-3 existed in a unimolecular form in a solution of 200 mM NaCl and that the
intermediate with g5p contained one I-3 strand with a free
3‘ end. The order of addition of the primer and g5p to I-3
was not important (data not shown), further suggesting that
the primer-annealing and g5p-binding sites on I-3 were not
overlapping and that about 16 nucleotides at the 3‘ end of I-3 were not involved in g5p binding to form the initiation
complex. In other experiments, mixtures of I-3 and various
concentrations of the 3‘ primer were pretreated by heating
to 95 °C and slow cooling to room temperature. The results
of these experiments (not shown) were essentially identical
to lanes 1−4 of Figure <xref rid="bi010109zf00004"></xref>A. Therefore, it was unlikely that
two or more I-3 strands folded asymmetrically so that only
one 3‘ end was accessible. Finally, gel electrophoresis of
serial dilutions of I-3 provided additional evidence that I-3
existed in a unimolecular form at both low and high sodium
concentrations. As shown in Figure <xref rid="bi010109zf00004"></xref>B, when diluted over
a 250-fold concentration range of 4 to 0.016 μM in either
10 or 200 mM NaCl, I-3 migrated as a single band in a native
gel.
</p><p>The molar ratio of protein to DNA in the intermediate
band 2 complex was determined by extraction of the band 2
complex, performing SDS−PAGE, and quantitating the
amount of stained protein and labeled I-3. Results are
tabulated in Table <xref rid="bi010109zt00004"></xref>. Together with the primer-annealing data,
these data revealed that the initiation complex consisted of
one I-3 strand and about three g5p dimers (six monomers).
In contrast, the saturation complex, formed at 10 or 200 mM
NaCl, had a stoichiometry that averaged about 15 g5p
monomers per I-3 strand (Table <xref rid="bi010109zt00004"></xref>), consistent with a g5p
binding mode of <italic toggle="yes">n</italic> = 4.
<table-wrap id="bi010109zt00004" position="float" orientation="portrait"><label>4</label><caption><p>Protein/DNA Stoichiometries of the Initiation and Saturation Complexes</p></caption><oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="4"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:colspec colnum="2" colname="2"></oasis:colspec><oasis:colspec colnum="3" colname="3"></oasis:colspec><oasis:colspec colnum="4" colname="4"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry namest="1" nameend="1"></oasis:entry><oasis:entry namest="2" nameend="2">saturation
complex
(10 mM NaCl)</oasis:entry><oasis:entry namest="3" nameend="3">initiation
complex
(200 mM NaCl)</oasis:entry><oasis:entry namest="4" nameend="4">saturation
complex
(200 mM NaCl)
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">g5p monomer
per I-3 strand<italic toggle="yes"><sup>a</sup></italic><sup></sup></oasis:entry><oasis:entry colname="2">15.5 ± 0.6
</oasis:entry><oasis:entry colname="3">6.4 ± 1.2
</oasis:entry><oasis:entry colname="4">14.8 ± 2.8</oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic><sup></sup> Data are shown as mean ± SD from at least three measurements.</p></table-wrap-foot></table-wrap></p><p><italic toggle="yes">The g5p-Binding Sites within the Initiation Complex.</italic> The
above results showed that g5p did not saturate the whole
I-3 sequence within the initiation complex. Nuclease S1
footprinting was used to investigate whether g5p bound
directly to the central G-rich region of the selected sequence
or whether the g5p bound to and protected the 5‘ and 3‘
primer ends of some type of folded I-3 structure that was
stabilized by the G-rich center. To avoid interference by the
band 1 complex, conditions for complex formation were
chosen such that the initiation complex was formed but the
saturated complex was not formed, i.e., the conditions were
the same as in lane 2 of Figure <xref rid="bi010109zf00001"></xref>B. Results shown in Figure
<xref rid="bi010109zf00005"></xref> established that the dominant g5p protection was directly
within the central G-rich region, extending from nucleotide
17 to 42. With the exception of nucleotide 35, the most
highly protected nucleotides were all G's and included two
G's in the 3‘ region (Figure <xref rid="bi010109zf00005"></xref>, horizontal bars).
<fig id="bi010109zf00005" position="float" orientation="portrait"><label>5</label><caption><p>Quantitative nuclease S1 footprinting. The 5‘ end<sup>32</sup>P-labeled I-3 (1 μM) was preincubated with or without 1.4 μM g5p
in 200 mM NaCl at 37 °C for 15 min. After treatment with nuclease
S1 for 1 min, the mixtures were resolved in 12% denaturing
polyacrylamide gels. Quantitative analysis was carried out and the
percent protection was plotted as a function of base position of
I-3. (See Experimental Procedures for details.) The percent protection is from the average of three independent experiments and
standard deviations are shown. The horizontal bars on the top
indicate nucleotides that were protected by at least half of the
maximum protection (which was 47%).</p></caption><graphic xlink:href="bi010109zf00005.tif" position="float" orientation="portrait"></graphic></fig></p><p><italic toggle="yes">End Boundaries of I-3 in the Initiation Complex.</italic> Nuclease
S1 footprinting (Figure <xref rid="bi010109zf00005"></xref>) suggested that the variable region
of I-3 contained the actual sites bound by g5p to form the
initiation complex. However, protection from nuclease S1
digestion could partially be an indirect effect of g5p binding.
Therefore, the sequence boundaries of the variable region
that were required for formation of the initiation complex
were more exactly defined. To determine the 3‘-end boundary, I-3 was <sup>32</sup>P-labeled at the 5‘ end and partially digested
by nuclease S1 to generate fragments of variable lengths that
extended from a fixed, labeled 5‘ end. When incubated at
200 mM NaCl with decreasing concentrations of g5p,
fragments that had reduced g5p-binding affinity were not
bound and could not be recovered by filter binding (see
Experimental Procedures). The left lane of Figure <xref rid="bi010109zf00006"></xref>A shows
that, as expected, a large range of sequence lengths, including
those that were truncated into the variable region, were
isolated at saturating concentrations of g5p. At lower
concentrations of g5p at which formation of the initiation
complex dominated, the lengths of DNA that were isolated
(and that could be detected) were longer, representing the
loss of successive nucleotides from the 3‘ unlabeled end. A
discrete boundary was identified, between nucleotides T41
and C42 at 7 μM g5p, or between C42 and A43 at lower
g5p concentrations, that represented the 3‘ end of the
sequence needed to form the initiation complex. This
boundary was within a nucleotide of the junction between
the variable and 3‘ primer region, which was between C42
and A43. The clear identification of this 3‘ boundary
indicated that G47 and G48, which were relatively protected
in the nuclease S1 footprinting experiments (Figure <xref rid="bi010109zf00005"></xref>), were
not part of the actual binding site and were possibly indirectly
protected by the bound g5p.
<fig id="bi010109zf00006" position="float" orientation="portrait"><label>6</label><caption><p>End boundaries of the I-3 sequence in the initiation complex with g5p. I-3 was labeled (A) at the 5‘ end for determining the 3‘-end boundary, and (B) at the 3‘ end for determining the 5‘-end boundary. Labeled I-3 was partially digested by nuclease S1 and incubated with various concentrations of g5p as shown on the figure. The fragments that could form complexes with g5p were selected by membrane filtration and resolved on 12% denaturing polyacrylamide gels, as described in Experimental Procedures. Bands were assigned, and the variable and two primer regions were identified and are marked on the figure. The nucleotide marking a given band is the last nucleotide (farthest from the labeled end) that is on that fragment.</p></caption><graphic xlink:href="bi010109zf00006.tif" position="float" orientation="portrait"></graphic></fig></p><p>In the reverse experiment, I-3 was labeled at the 3‘ end to
determine the 5‘-end boundary. Figure <xref rid="bi010109zf00006"></xref>B shows that there
was also a discrete 5‘ boundary, between T16 and G17 at
3.5−14 μM g5p, or between T16 and T15 at 0.9 μM g5p,
where the former was at the junction between the variable
and 5‘ primer region. Therefore, the boundary experiments
confirmed that g5p bound directly to the variable region to
form the initiation complex and that the minimum motif for
forming an initiation complex with g5p essentially encompassed the entire variable sequence between the junctions
with the two primer regions.
</p></sec><sec id="d7e959"><title>Discussion</title><p><italic toggle="yes">SELEX of a Cooperative ssDNA Binding Protein.</italic> SELEX
has been successfully used to isolate, from an ssDNA library
of 58-mers (PV-58), those sequences that bind g5p with high-affinity under physiologically relevant conditions (200 mM
NaCl, 37 °C, and pH 7.4). The variable region of PV-58
molecules consisted of the central 26 nucleotides. The
selection of sequences by g5p was based on the formation
of a discrete electrophoretic band of apparently saturated
g5p•ssDNA complexes during competitive binding of PV-58 sequences to a limited amount of g5p. Owing to its high
cooperativity, g5p will usually bind to and saturate every
site on most PV-58 sequences, including the two flanking
16-mer constant sequences. In terms of applying SELEX,
this behavior is the major difference between cooperative
DNA binding proteins (such as g5p) and specific DNA-binding proteins. The latter type of protein generally binds
with one protein molecule per DNA strand, and the binding
site is usually selected from and located within the variable
region, although part of the primer sequence can be involved
in binding (<italic toggle="yes"><xref rid="bi010109zb00030" ref-type="bibr"></xref></italic>). Given the fact that the binding site for g5p
is relatively small (three to four nucleotides per g5p
monomer; <italic toggle="yes">6</italic>), the stretch of 26 selectable nucleotides in the
central, variable region of PV-58 was apparently long enough
to create a structured site that favored the initial binding of
several g5p dimers. Thus, the cooperative binding nature of
g5p, which led to subsequent saturation of the constant
sequences, did not prevent the selection of sequences that
bound with high affinity.
</p><p><italic toggle="yes">Characterization of an I-3 Selected Sequence and an
Initiation Complex.</italic> After eight rounds of selection, most
SELEX-derived sequences for g5p binding were G-rich and
had one or more similar motifs such as CPuGGPy, TPuGGGPy, and PyPuPuGGGPy (see Table <xref rid="bi010109zt00001"></xref>). This was surprising
because g5p is well-known to bind with higher affinity to
synthetic oligonucleotides that are pyrimidine-rich than to
those that are purine-rich (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi010109zb00009" ref-type="bibr"></xref>, <xref rid="bi010109zb00010" ref-type="bibr"></xref></named-content></italic>). However, analyses of the
cloned sequences from intermediate rounds of selection
showed that the selected G-rich sequences were finally
selected from larger pools of intermediate sequences that
were indeed pyrimidine rich (Table <xref rid="bi010109zt00002"></xref>). In addition, under
the selection conditions of 200 mM NaCl and 37 °C, the
g5p-binding affinities for ssDNA sequences that are purine-rich are weakened, because the binding to purine-rich
sequences is largely driven by ion release and ionic interactions. Nevertheless, for a predominant selected sequence, I-3,
the g5p binding affinity was increased by over 2 orders of
magnitude as compared with its averaged binding affinity
to the original mixture of PV-58 sequences (Table <xref rid="bi010109zt00003"></xref>).
</p><p>Titrations of I-3 with g5p were performed and monitored
independently by EMSA (Figure <xref rid="bi010109zf00001"></xref>) and by CD spectroscopy
(Figure <xref rid="bi010109zf00003"></xref>). The results were consistent in showing that, prior
to saturation, an intermediate initiation complex was formed
at 200 mM but not at 10 mM NaCl. The formation of this
initiation complex appeared to be a key factor that resulted
in a higher binding affinity of g5p for I-3 at 200 mM than
at 10 mM NaCl (Table <xref rid="bi010109zt00003"></xref>). CD spectroscopy also suggested
that, at 200 mM NaCl, free I-3 formed a structure, probably
involving a G-quadruplex, to which, according to the data
in Table <xref rid="bi010109zt00004"></xref>, about three g5p dimers bound to form the
initiation complex. The initiation complex was apparently
formed in an all-or-none fashion, because there were no
intermediate bands between the free I-3 and band 2 on
agarose gels (Figure <xref rid="bi010109zf00001"></xref>). That is, it appeared that all three
g5p simultaneously bound to form a core initiation complex
for further saturation. Moreover, CD spectroscopy showed
that the I-3 structure was maintained largely intact within
the saturated complex.
</p><p>The binding site for the g5p dimers that form an initiation
complex was essentially identical to the 26-mer, central
G-rich selected region of I-3 (Figures <xref rid="bi010109zf00003"></xref>, <xref rid="bi010109zf00005"></xref>, and 6). Our
knowledge about the initiation structure is illustrated in
Figure <xref rid="bi010109zf00007"></xref>. One I-3 molecule is involved. The central 26-mer
(nucleotides 17−42) is G-rich, and this sequence has the
potential to be folded into one of several unimolecular
G-quadruplexes, such as the chair form described for the
thrombin aptamer in the presence of K<sup>+</sup> (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi010109zb00028" ref-type="bibr"></xref>, <xref rid="bi010109zb00029" ref-type="bibr"></xref></named-content></italic>). Although
the structure of I-3 in the presence of Na<sup>+</sup> is not known, it
is not likely to be a simple chair G-quadruplex (Figure <xref rid="bi010109zf00003"></xref>).
Three g5p dimers bind directly to this central region to form
an initiation complex that migrates as “band 2” in EMSA
experiments. We assume that the 3‘ and 5‘ primer ends are
juxtaposed in an antiparallel fashion for subsequent saturation
by additional g5p dimers (which have rotationally symmetric
binding sites) to form the “band 1” complex. A key point is
that the putative G-quadruplex structure may not only
stabilize a desirable template for binding, but the structure
itself appears to be the actual initial g5p binding site.
<fig id="bi010109zf00007" position="float" orientation="portrait"><label>7</label><caption><p>Schematic of the alignment of the I-3 sequence when complexed with g5p at 200 mM NaCl. There are two major regions to the structure. Region (1) consists of nucleotides 16−42 and is essentially identical with the selected G-rich variable region. This region may form a structure by folding into a unimolecular G-quadruplex. About three g5p dimers bind directly to this region to form the band 2 initiation complex detected by EMSA. The shaded bar highlights the nucleotides that are at the 3‘- and 5‘-end boundaries. Region (2) consists of the 5‘ and 3‘ primer sequences. These presumably are oriented in an antiparallel fashion and provide binding sites that are subsequently saturated to form the band 1 complex in EMSA experiments.</p></caption><graphic xlink:href="bi010109zf00007.tif" position="float" orientation="portrait"></graphic></fig></p><p><italic toggle="yes">Biological Relevance.</italic> The abilities of Ff g5p to form both
a cooperatively saturated complex through nonspecific binding and an unsaturated complex through specific binding
correspond to the biological functions of g5p in sequestering
the viral genome and regulating the translation of viral
mRNAs, respectively. The g5p is known to saturate the
nascent Ff ssDNA genome for phage genome packaging (<italic toggle="yes"><xref rid="bi010109zb00001" ref-type="bibr"></xref></italic>).
In this sense, g5p acts as a non-sequence-specific ssDNA
binding protein and binds in a cooperative manner, even
though the nucleation process is still unknown. On the other
hand, g5p is also involved in translational regulation of some
viral mRNAs, such as gene 2 mRNA, by binding to a specific
sequence in the 5‘-end untranslated region of the mRNA (see
below). The behavior of g5p binding to the SELEX-derived
sequences can provide insight into how cooperative interactions are initiated under physiological conditions and into
the types of sequences and/or structures of DNA or RNA
that might be specific binding sites.
</p><p>In addition to the in vitro-selected I-3 sequence, a naturally
occurring sequence has also been found to form an unsaturated intermediate complex with g5p. Michel and Zinder (<italic toggle="yes"><xref rid="bi010109zb00017" ref-type="bibr"></xref></italic>)
showed that the first 16 nucleotides (5‘-GUUUUUGGGGCUUUUC-3‘) of the Ff gene 2 mRNA leader sequence is
required for g5p-mediated translational repression of this
mRNA and that an RNA (208−211 bases in length)
containing this leader sequence forms an intermediate
complex in gel electrophoresis before a saturated complex
is formed (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi010109zb00017" ref-type="bibr"></xref>, <xref rid="bi010109zb00018" ref-type="bibr"></xref></named-content></italic>). The g5p binds to this RNA with a 10-fold higher affinity than to the control RNA. How g5p binds
specifically to this sequence is still not clear, but the apparent
lack of structure in this region was originally thought to be
a dominant factor (<italic toggle="yes"><xref rid="bi010109zb00014" ref-type="bibr"></xref></italic>). However, a likely tetraplex structure
with a central block of G-quartets has recently been shown
to form with four strands of the gene 2 leader sequence or
with its DNA analogue (<italic toggle="yes"><xref rid="bi010109zb00044" ref-type="bibr"></xref></italic>). Kneale and co-workers also
demonstrated preferential binding of g5p to this structure
(<italic toggle="yes"><xref rid="bi010109zb00045" ref-type="bibr"></xref></italic>). They propose that tails of four antiparallel strands, held
together by G-quartets, are separated by the right distance
to occupy the two symmetry-related binding sites on a g5p
dimer and thus to initiate g5p binding (<italic toggle="yes"><xref rid="bi010109zb00045" ref-type="bibr"></xref></italic>). In their model,
g5p does not bind directly to the G-quartet structure.
</p><p>Our data with I-3 provides support for the idea that g5p
prefers to bind to a structured sequence, but in a somewhat
different manner than that proposed by Oliver et al. (<italic toggle="yes"><xref rid="bi010109zb00045" ref-type="bibr"></xref></italic>) for
the gene 2 leader sequence. The I-3 structure is formed with
only one strand (Figure <xref rid="bi010109zf00004"></xref>). Moreover, our data show that
G<sub>4</sub> blocks play a direct role in the high affinity binding
(Figures <xref rid="bi010109zf00005"></xref> and <xref rid="bi010109zf00006"></xref>). The conformation of I-3 in the initiation
complex presumably orients two antiparallel strands (the 5‘
and 3‘ primer strands; see Figure <xref rid="bi010109zf00007"></xref>) to form additional g5p
dimer binding sites that are subsequently saturated; however,
these are not the first sites to be bound by g5p, as might be
expected by analogy with the structure of the gene 2 leader
sequence.
</p><p>It remains to be seen whether the (g5p)<sub>3</sub>·I-3 initiation
complex has a biological counterpart or whether such a
complex would be formed with RNA. It is relevant to point
out that an intrastrand interaction could occur between the
gene 2 leader 16-mer and nearby sequences of the gene 2
mRNA to form a secondary structure. Specifically, in the
gene 2 leader sequence there is another G<sub>4</sub> block (part of
the Shine-Dalgarno sequence) that is downstream and
separated from the G<sub>4</sub> block in the leader 16-mer by only
18 bases (<italic toggle="yes"><xref rid="bi010109zb00046" ref-type="bibr"></xref></italic>). Interaction of these two G<sub>4</sub> blocks through
antiparallel G:G pairing could plausibly form a structure to
facilitate g5p binding. On the other hand, we found that the
specific SELEX-selected motifs (such as CAGGPy and
NGGGN; see Table <xref rid="bi010109zt00001"></xref>) occurred less frequently in the Ff
genome (60 times) than in a random sequence of 6000
nucleotides (88 times) and that the motifs did not have an
unusual distribution. Therefore, the biological relevance of
these specific motifs within the Ff genome remains uncertain.
</p><p><italic toggle="yes">Protein Binding to G-rich Sequences.</italic> G-quartets are also
found in other SELEX-derived aptamers for thrombin (<italic toggle="yes"><xref rid="bi010109zb00027" ref-type="bibr"></xref></italic>),
elastase (<italic toggle="yes"><xref rid="bi010109zb00047" ref-type="bibr"></xref></italic>), and IgE (<italic toggle="yes"><xref rid="bi010109zb00048" ref-type="bibr"></xref></italic>). G-rich telomere sequences are
bound by α and β telomere-binding proteins and by the
Cdc13p telomerase-loading protein (<italic toggle="yes">39</italic>,<italic toggle="yes"> 49−51</italic>). Human
DNA topoisomerases I and II interact with G-quartet
structures (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi010109zb00052" ref-type="bibr"></xref>, <xref rid="bi010109zb00053" ref-type="bibr"></xref></named-content></italic>) and other proteins that interact with
G-quartets have been reviewed (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi010109zb00052" ref-type="bibr"></xref>, <xref rid="bi010109zb00054" ref-type="bibr"></xref></named-content></italic>). Loops on intrastrand
G-quartet or hairpin structures can be the sites for target
binding (<italic toggle="yes"><xref rid="bi010109zb00022" ref-type="bibr"></xref></italic>). In the case of I-3, some of the loop nucleotides
may just be responsible for connecting g5p-binding sites and
positioning them in an appropriate three-dimensional conformation, since they appear not to be protected by g5p from
nuclease digestion (Figure <xref rid="bi010109zf00005"></xref>). Nevertheless, the many
examples of proteins that bind to G-rich sequences suggest
that G-rich motifs selected by the g5p may have features
that are recognized by other proteins, and the study of
g5p•ssDNA initiation complexes could be of general importance for understanding the binding, or initiation of
cooperative binding, of other single-stranded DNA-binding
proteins.
</p></sec></body><back><ack><title>Acknowledgments</title><p>We are grateful to Dr. Tung-Chung Mou (University of
Texas at Dallas) for generously providing purified wild-type
g5p and to Dr. Andy Peek of Cytoclonal Pharmaceutics, Inc.
(Dallas) for generating a random DNA sequence. We have
appreciated the advice and encouragement of Drs. Larry Gold
(Department of Molecular, Cellular, and Developmental
Biology, University of Colorado, Boulder, CO), Dennis L.
Miller (University of Texas at Dallas), and Thomas C.
Terwilliger (Los Alamos National Laboratory, Los Alamos,
NM) throughout the course of this work.
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Department ofMolecular and Cell Biology, Mail Stop FO 3.1, The University of Texasat Dallas, Box 830688, Richardson, TX 75083-0688; (972) 883-2513;FAX (972) 883-2409; e-mail: dongray@utdallas.edu.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="research-article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre><originInfo><publisher>American Chemical Society</publisher><dateCreated encoding="w3cdtf">2001-07-12</dateCreated><dateIssued encoding="w3cdtf">2001-08-07</dateIssued><copyrightDate encoding="w3cdtf">2001</copyrightDate></originInfo><note type="footnote" ID="bi010109zAF2"> This work was performed by J.-D.W. in partial fulfillment of the requirements for the Ph.D. degree in the Department of Molecular and Cell Biology, The University of Texas at Dallas. Support was provided by grants from the Robert A. Welch Foundation (AT-503) and the Texas Advanced Technology Program (009741-0021-1999).</note><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract>The Ff gene 5 protein (g5p) is a cooperative ssDNA-binding protein. SELEX was used to identify DNA sequences favorable for g5p binding at physiological ionic strength (200 mM NaCl) and 37 °C. Sequences were selected from a library of 58-mers that contained a central variable segment of 26 nucleotides. DNA sequences selected after eight rounds of SELEX were mostly G-rich, with multiple copies of CPuGGPy, TPuGGGPy, and/or PyPuPuGGGPy motifs. This was unexpected, since g5p has higher binding affinities for polypyrimidine than for polypurine sequences. The most recurrent G-rich sequence, named I-3, was found to have g5p-binding properties that were correlated with a structural transition. At 10 mM NaCl, I-3 existed in a single-stranded form that was saturated by g5p in an all-or-none fashion. At 200 mM NaCl, I-3 existed in a structured form that showed CD spectral features of G-quadruplexes. The g5p binding affinity for this structured form of I-3 was >100-fold higher than for the single-stranded form. Moreover, the structured I-3 was saturated by g5p in two steps, the first of which was the formation of an apparent initiation complex consisting of one I-3 strand and about three g5p dimers. Nuclease S1 footprinting and other experiments showed that g5p molecules in the initiation complex at 200 mM NaCl were bound directly to the G-rich variable segment and that the structure of I-3 was retained after saturation by g5p. Thus, G-rich motifs may form structures favorable for initiation of g5p binding and also provide the actual g5p-binding sites.</abstract><note type="footnote" ID="bi010109zAF2"> This work was performed by J.-D.W. in partial fulfillment of the requirements for the Ph.D. degree in the Department of Molecular and Cell Biology, The University of Texas at Dallas. Support was provided by grants from the Robert A. 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Struct. 1994 23 703 730 10.1146/annurev.bb.23.060194.003415</note><identifier type="doi">10.1146/annurev.bb.23.060194.003415</identifier><part><date>1994</date><detail type="volume"><caption>vol.</caption><number>23</number></detail><extent unit="pages"><start>703</start><end>730</end></extent></part></relatedItem><relatedItem type="references" ID="bi010109zn00001" displayLabel="bibbi010109zn00001"><titleInfo><title>Abbreviations: aptamer, a nucleic acid sequence selected to have high affinity for a protein or other substance; CD, circular dichroism; EMSA, electrophoretic mobility shift assay; Ff phages, three closely related filamentous viruses f1, fd, and M13 that specifically infect F+strains ofEscherichia coli; g5p, gene 5 protein; Kω, the intrinsic binding constant (K) times a cooperativity factor (ω); PCR, polymerase chain reaction; P/N, the [protein monomer]/[nucleotide] molar ratio; SDS−PAGE, sodium dodecyl sulfate−polyacrylamide gel electrophoresis; SELEX, Systematic Evolution of Ligands by Exponential enrichment; ssDNA, single-stranded DNA; TAE buffer, 40 mM Tris-acetate, pH 8.3, 1 mM EDTA; TBE buffer, 90 mM Tris-borate, pH 8.3, 2 mM EDTA; TE buffer, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA.</title></titleInfo><note type="content-in-line">Abbreviations: aptamer, a nucleic acid sequence selected to have high affinity for a protein or other substance; CD, circular dichroism; EMSA, electrophoretic mobility shift assay; Ff phages, three closely related filamentous viruses f1, fd, and M13 that specifically infect F+ strains of Escherichia coli; g5p, gene 5 protein; Kω, the intrinsic binding constant (K) times a cooperativity factor (ω); PCR, polymerase chain reaction; P/N, the [protein monomer]/[nucleotide] molar ratio; SDS−PAGE, sodium dodecyl sulfate−polyacrylamide gel electrophoresis; SELEX, Systematic Evolution of Ligands by Exponential enrichment; ssDNA, single-stranded DNA; TAE buffer, 40 mM Tris-acetate, pH 8.3, 1 mM EDTA; TBE buffer, 90 mM Tris-borate, pH 8.3, 2 mM EDTA; TE buffer, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA.</note></relatedItem><identifier type="istex">373A64E897640F4570DDCACBFFC388C995F2720C</identifier><identifier type="ark">ark:/67375/TPS-KD58MTD9-V</identifier><identifier type="DOI">10.1021/bi010109z</identifier><accessCondition type="use and reproduction" contentType="restricted">Copyright © 2001 American Chemical Society</accessCondition><recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-X5HBJWF8-J">acs</recordContentSource><recordOrigin>Converted from (version 1.2.10) to MODS version 3.6.</recordOrigin><recordCreationDate encoding="w3cdtf">2020-04-10</recordCreationDate></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-KD58MTD9-V/record.json</uri></json:item></metadata><annexes><json:item><extension>tiff</extension><original>true</original><mimetype>image/tiff</mimetype><uri>https://api.istex.fr/document/373A64E897640F4570DDCACBFFC388C995F2720C/annexes/tiff</uri></json:item></annexes><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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