https://www.synbiocyc.org/wiki/index.php?title=CH391L/S14/Spinach_RNA&feed=atom&action=historyCH391L/S14/Spinach RNA - Revision history2024-03-28T11:34:21ZRevision history for this page on the wikiMediaWiki 1.21.1https://www.synbiocyc.org/wiki/index.php?title=CH391L/S14/Spinach_RNA&diff=96578&oldid=prevDst465 at 15:46, 17 March 20142014-03-17T15:46:06Z<p></p>
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<tr><td class='diff-marker'>−</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>'''Spinach''' is an RNA aptamer which binds to the small molecule 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), which is structurally similar to the fluorophore in [http://www.synbiocyc.org/wiki/index.php/CH391L/S14/GFP green fluorescent protein] (GFP), hydroxybenzylidene imidazolinone (HBI).  Upon binding, the aptamer-small molecule complex becomes highly fluorescent, similar to GFP fluorescence.  A variety of HBI derivatives with corresponding aptamers have been developed which fluoresce upon binding and provide a range of excitation and emission wavelengths.  Spinach allows for high resolution monitoring of mRNA transcripts, providing information on transcription rates, and localization of mRNAs ''in vivo''. Spinach was originally reported in Science Magazine in July 2011, and has since been cited by [http://scholar.google.com/scholar?rlz=1C1CHFX_enUS569US569&espv=210&es_sm=93&um=1&ie=UTF-8&lr=&cites=15840443076454798134 120 articles].</div></td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>'''Spinach''' is an RNA aptamer which binds to the small molecule 3,5-difluoro-4-hydroxybenzylidene imidazolinone (<ins class="diffchange diffchange-inline">'''</ins>DFHBI<ins class="diffchange diffchange-inline">'''</ins>), which is structurally similar to the fluorophore in [http://www.synbiocyc.org/wiki/index.php/CH391L/S14/GFP green fluorescent protein] (GFP), hydroxybenzylidene imidazolinone (HBI).  Upon binding, the aptamer-small molecule complex becomes highly fluorescent, similar to GFP fluorescence.  A variety of HBI derivatives with corresponding aptamers have been developed which fluoresce upon binding and provide a range of excitation and emission wavelengths.  Spinach allows for high resolution monitoring of mRNA transcripts, providing information on transcription rates, and localization of mRNAs ''in vivo''. Spinach was originally reported in Science Magazine in July 2011, and has since been cited by [http://scholar.google.com/scholar?rlz=1C1CHFX_enUS569US569&espv=210&es_sm=93&um=1&ie=UTF-8&lr=&cites=15840443076454798134 120 articles].</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>===Theory===</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>===Theory===</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>The natural fluorophore of GFP is formed through the auto-cyclization of three adjacent amino acids, Ser65, Tyr66, and Gly67, which react under oxidizing conditions to form hydroxybenzlidene imidazolinone (HBI).  [[File:HBI and GFP.png|thumb|200px|right| A comparison of the GFP fluorophore after autocyclization and one of the HBI derivatives used by the Jaffrey Lab <cite>Jaffrey2011</cite>]] HBI itself is not significantly fluorescent, but becomes fluorescent when it is vibration restricted<cite>Meech2009</cite>.  Side chain contacts made with HBI in GFP prevent intramolecular motions, which restrict the energy dissipation pathways available for HBI after excitation by a photon, this restriction leaves fluorescence as the most readily available and common pathway for relaxation.  It was hypothesized by the [http://www.jaffreylab.org/Pages/default.aspx Jaffrey lab] that restricting motions of HBI using an RNA aptamer instead of proteinogenic amino acids could result in a similar fluorescence to GFP<cite>Jaffrey2011</cite>.  An aptamer is a small oligonucleotide which binds to a target molecule, they are identified from pools of random oligonucleotides through affinity selection<cite>Ellington1990</cite>. The group suspected that a modular, fluorescent RNA aptamer would facilitate RNA studies and RNA technologies, similar to the effect GFP has had on protein biochemistry and biotechnology.</div></td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>The natural fluorophore of GFP is formed through the auto-cyclization of three adjacent amino acids, Ser65, Tyr66, and Gly67, which react under oxidizing conditions to form hydroxybenzlidene imidazolinone (<ins class="diffchange diffchange-inline">'''</ins>HBI<ins class="diffchange diffchange-inline">'''</ins>).  [[File:HBI and GFP.png|thumb|200px|right| A comparison of the GFP fluorophore after autocyclization and one of the HBI derivatives used by the Jaffrey Lab <cite>Jaffrey2011</cite>]] HBI itself is not significantly fluorescent, but becomes fluorescent when it is vibration restricted<cite>Meech2009</cite>.  Side chain contacts made with HBI in GFP prevent intramolecular motions, which restrict the energy dissipation pathways available for HBI after excitation by a photon, this restriction leaves fluorescence as the most readily available and common pathway for relaxation.  It was hypothesized by the [http://www.jaffreylab.org/Pages/default.aspx Jaffrey lab] that restricting motions of HBI using an RNA aptamer instead of proteinogenic amino acids could result in a similar fluorescence to GFP<cite>Jaffrey2011</cite>.  An aptamer is a small oligonucleotide which binds to a target molecule, they are identified from pools of random oligonucleotides through affinity selection<cite>Ellington1990</cite>. The group suspected that a modular, fluorescent RNA aptamer would facilitate RNA studies and RNA technologies, similar to the effect GFP has had on protein biochemistry and biotechnology.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>===Development and Physical Properties===</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>===Development and Physical Properties===</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>===Uses===</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>===Uses===</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>Spinach is useful as a modular appendage to RNA transcripts in the cell.  The addition of the 60 bp spinach aptamer to the end of coding and noncoding transcripts in cells has been shown to allow for monitoring of RNA localization in vivo.  The Jaffrey lab demonstrated this in the orginal spinach paper by appending spinach to the 3’ end of 5S ribosomal RNA<cite>Jaffrey2011</cite>.  By growing human cells with this modified gene in the presence of DFHBI, similar fluorescent patterns were seen as other experiments who had monitored 5S localization.  Additionally, when placed under stress, the cell responded by forming RNA granules colocalized with T-cell intracellular antigen-1-related protein, as was expected of the stress conditions.</div></td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>Spinach is useful as a modular appendage to RNA transcripts in the cell.  The addition of the 60 bp spinach aptamer to the <ins class="diffchange diffchange-inline">5' or 3' </ins>end of coding and noncoding transcripts in cells has been shown to allow for monitoring of RNA localization <ins class="diffchange diffchange-inline">''</ins>in vivo<ins class="diffchange diffchange-inline">''</ins>.  The Jaffrey lab demonstrated this in the orginal spinach paper by appending spinach to the 3’ end of 5S ribosomal RNA<cite>Jaffrey2011</cite>.  By growing human cells with this modified gene in the presence of DFHBI, similar fluorescent patterns were seen as other experiments who had monitored 5S localization.  Additionally, when placed under stress, the cell responded by forming RNA granules colocalized with T-cell intracellular antigen-1-related protein, as was expected of the stress conditions.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>Jaffrey was also able to develop a spinach variant which is capable of detecting a second molecule, specifically S-Adenosyl-Methionine (SAM)<cite>Jaffrey2012</cite>.  The aptamer binds to both DFHBI and SAM, and only when bound to both small molecules does the complex fluoresce.  This technology could be expanded to a variety of small molecules, and used as a sensitive detection method.</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>Jaffrey was also able to develop a spinach variant which is capable of detecting a second molecule, specifically S-Adenosyl-Methionine (SAM)<cite>Jaffrey2012</cite>.  The aptamer binds to both DFHBI and SAM, and only when bound to both small molecules does the complex fluoresce.  This technology could be expanded to a variety of small molecules, and used as a sensitive detection method.</div></td></tr>
</table>Dst465https://www.synbiocyc.org/wiki/index.php?title=CH391L/S14/Spinach_RNA&diff=96462&oldid=prevDst465 at 18:16, 4 March 20142014-03-04T18:16:52Z<p></p>
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<td colspan='2' style="background-color: white; color:black; text-align: center;">Revision as of 18:16, 4 March 2014</td>
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<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>Individual sequences were screened for aptamers that accounted for the increased fluorescence observed, and the aptamer which gave the highest fluorescence was identified and named 13-2.  The Jaffrey lab then used truncations to determine a minimal aptamer [[File:Spinach derivatives.png|thumb|300px|right|A variety of aptamers against HBI derivatives results in a spectral range of fluoresence]]sequence, which resulted in an increase in quantum yield of fluorescence.  The final aptamer 13-2 is 60 nucleotides long, exhibited an emission peak of 529 nm, and an excitation peak of 398 nm.  Further selections allowed for tuning to a range of spectral properties, providing different color fluorescent molecules all using DMHBI as the substrate<cite>Jaffrey2011</cite>.   </div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>Individual sequences were screened for aptamers that accounted for the increased fluorescence observed, and the aptamer which gave the highest fluorescence was identified and named 13-2.  The Jaffrey lab then used truncations to determine a minimal aptamer [[File:Spinach derivatives.png|thumb|300px|right|A variety of aptamers against HBI derivatives results in a spectral range of fluoresence]]sequence, which resulted in an increase in quantum yield of fluorescence.  The final aptamer 13-2 is 60 nucleotides long, exhibited an emission peak of 529 nm, and an excitation peak of 398 nm.  Further selections allowed for tuning to a range of spectral properties, providing different color fluorescent molecules all using DMHBI as the substrate<cite>Jaffrey2011</cite>.   </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'>−</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>The quantum yield of GFP fluorescence was greatly improved by modification of the protein and screening, which lead to enhanced GFP, or EGFP.  The EGFP fluorophore is most often in the phenolate form  while wild type GFP is most commonly in the phonol form<cite>Tsien1998</cite><cite>Heim1995</cite>, this phenolate [[File:Phenolate DFHBI.png|100 px|right|The HBI derivative DFHBI in the phenolate form, which is more fluorescent]]from is suspected to be the reason for EGFPs significant improvement in fluorescence.  Following that logic, the Jaffrey group set out to copy this by modifying the HBI derivative used in an attempt to obtain the phenolate form of HBI seen in EGFP.  The group swapped the methoxy-groups on the phenol ring to electron withdrawing fluorines, forming difluoro-HBI (DFHBI).  Selection for an aptamer against DFHBI resulted in an aptamer-DFHBI complex with significantly improved quantum yield over 13-2-DMHBI complex fluorescence.  The resulting complex shows 53% of the molar brightness of EGFP.  This aptamer, 24-2, was given the name “spinach”<cite>Jaffrey2011</cite>.</div></td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>The quantum yield of GFP fluorescence was greatly improved by modification of the protein and screening, which lead to enhanced GFP, or EGFP.  The EGFP fluorophore is most often in the phenolate form  while wild type GFP is most commonly in the phonol form<cite>Tsien1998</cite><cite>Heim1995</cite>, this phenolate [[File:Phenolate DFHBI.png<ins class="diffchange diffchange-inline">|thumb</ins>|100 px|right|The HBI derivative DFHBI in the phenolate form, which is more fluorescent]]from is suspected to be the reason for EGFPs significant improvement in fluorescence.  Following that logic, the Jaffrey group set out to copy this by modifying the HBI derivative used in an attempt to obtain the phenolate form of HBI seen in EGFP.  The group swapped the methoxy-groups on the phenol ring to electron withdrawing fluorines, forming difluoro-HBI (DFHBI).  Selection for an aptamer against DFHBI resulted in an aptamer-DFHBI complex with significantly improved quantum yield over 13-2-DMHBI complex fluorescence.  The resulting complex shows 53% of the molar brightness of EGFP.  This aptamer, 24-2, was given the name “spinach”<cite>Jaffrey2011</cite>.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>===Uses===</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>===Uses===</div></td></tr>
</table>Dst465https://www.synbiocyc.org/wiki/index.php?title=CH391L/S14/Spinach_RNA&diff=96461&oldid=prevDst465 at 18:16, 4 March 20142014-03-04T18:16:35Z<p></p>
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<td colspan='2' style="background-color: white; color:black; text-align: center;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black; text-align: center;">Revision as of 18:16, 4 March 2014</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 7:</td>
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<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>The group synthesized HBI derivatives and tested their hypothesis, first by exploring the fluorescence of HBI derivative ''in vivo,'' to ensure that HBI was not fluorescent under normal cellular conditions<cite>Jaffrey2011</cite>.  No significant fluorescence was detected from the HBI derivatives, and they proceeded by selecting for aptamers against the HBI derivative 3,5-dimethoxy-4-hydroxybenzylidene imidazolinone (DMHBI) using SELEX (systematic evolution of ligands by exponential enrichment).  After five rounds of selection, an increase in fluorescence was detected, and each subsequent round of selection saw an increase in fluorescence, up to the tenth round of selection.</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>The group synthesized HBI derivatives and tested their hypothesis, first by exploring the fluorescence of HBI derivative ''in vivo,'' to ensure that HBI was not fluorescent under normal cellular conditions<cite>Jaffrey2011</cite>.  No significant fluorescence was detected from the HBI derivatives, and they proceeded by selecting for aptamers against the HBI derivative 3,5-dimethoxy-4-hydroxybenzylidene imidazolinone (DMHBI) using SELEX (systematic evolution of ligands by exponential enrichment).  After five rounds of selection, an increase in fluorescence was detected, and each subsequent round of selection saw an increase in fluorescence, up to the tenth round of selection.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>    </div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>    </div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>Individual sequences were screened for aptamers that accounted for the increased fluorescence observed, and the aptamer which gave the highest fluorescence was identified and named 13-2.  The Jaffrey lab then used truncations to determine a minimal aptamer [[File:Spinach derivatives.png|300px|right|A variety of aptamers against HBI derivatives results in a spectral range of fluoresence]]sequence, which resulted in an increase in quantum yield of fluorescence.  The final aptamer 13-2 is 60 nucleotides long, exhibited an emission peak of 529 nm, and an excitation peak of 398 nm.  Further selections allowed for tuning to a range of spectral properties, providing different color fluorescent molecules all using DMHBI as the substrate<cite>Jaffrey2011</cite>.   </div></td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>Individual sequences were screened for aptamers that accounted for the increased fluorescence observed, and the aptamer which gave the highest fluorescence was identified and named 13-2.  The Jaffrey lab then used truncations to determine a minimal aptamer [[File:Spinach derivatives.png<ins class="diffchange diffchange-inline">|thumb</ins>|300px|right|A variety of aptamers against HBI derivatives results in a spectral range of fluoresence]]sequence, which resulted in an increase in quantum yield of fluorescence.  The final aptamer 13-2 is 60 nucleotides long, exhibited an emission peak of 529 nm, and an excitation peak of 398 nm.  Further selections allowed for tuning to a range of spectral properties, providing different color fluorescent molecules all using DMHBI as the substrate<cite>Jaffrey2011</cite>.   </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>The quantum yield of GFP fluorescence was greatly improved by modification of the protein and screening, which lead to enhanced GFP, or EGFP.  The EGFP fluorophore is most often in the phenolate form  while wild type GFP is most commonly in the phonol form<cite>Tsien1998</cite><cite>Heim1995</cite>, this phenolate [[File:Phenolate DFHBI.png|100 px|right|The HBI derivative DFHBI in the phenolate form, which is more fluorescent]]from is suspected to be the reason for EGFPs significant improvement in fluorescence.  Following that logic, the Jaffrey group set out to copy this by modifying the HBI derivative used in an attempt to obtain the phenolate form of HBI seen in EGFP.  The group swapped the methoxy-groups on the phenol ring to electron withdrawing fluorines, forming difluoro-HBI (DFHBI).  Selection for an aptamer against DFHBI resulted in an aptamer-DFHBI complex with significantly improved quantum yield over 13-2-DMHBI complex fluorescence.  The resulting complex shows 53% of the molar brightness of EGFP.  This aptamer, 24-2, was given the name “spinach”<cite>Jaffrey2011</cite>.</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>The quantum yield of GFP fluorescence was greatly improved by modification of the protein and screening, which lead to enhanced GFP, or EGFP.  The EGFP fluorophore is most often in the phenolate form  while wild type GFP is most commonly in the phonol form<cite>Tsien1998</cite><cite>Heim1995</cite>, this phenolate [[File:Phenolate DFHBI.png|100 px|right|The HBI derivative DFHBI in the phenolate form, which is more fluorescent]]from is suspected to be the reason for EGFPs significant improvement in fluorescence.  Following that logic, the Jaffrey group set out to copy this by modifying the HBI derivative used in an attempt to obtain the phenolate form of HBI seen in EGFP.  The group swapped the methoxy-groups on the phenol ring to electron withdrawing fluorines, forming difluoro-HBI (DFHBI).  Selection for an aptamer against DFHBI resulted in an aptamer-DFHBI complex with significantly improved quantum yield over 13-2-DMHBI complex fluorescence.  The resulting complex shows 53% of the molar brightness of EGFP.  This aptamer, 24-2, was given the name “spinach”<cite>Jaffrey2011</cite>.</div></td></tr>
</table>Dst465https://www.synbiocyc.org/wiki/index.php?title=CH391L/S14/Spinach_RNA&diff=96460&oldid=prevDst465 at 18:16, 4 March 20142014-03-04T18:16:14Z<p></p>
<table class='diff diff-contentalign-left'>
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<td colspan='2' style="background-color: white; color:black; text-align: center;">Revision as of 18:16, 4 March 2014</td>
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<td colspan="2" class="diff-lineno">Line 2:</td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>===Theory===</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>===Theory===</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>The natural fluorophore of GFP is formed through the auto-cyclization of three adjacent amino acids, Ser65, Tyr66, and Gly67, which react under oxidizing conditions to form hydroxybenzlidene imidazolinone (HBI).  [[File:HBI and GFP.png|200px|right| A comparison of the GFP fluorophore after autocyclization and one of the HBI derivatives used by the Jaffrey Lab <cite>Jaffrey2011</cite>]] HBI itself is not significantly fluorescent, but becomes fluorescent when it is vibration restricted<cite>Meech2009</cite>.  Side chain contacts made with HBI in GFP prevent intramolecular motions, which restrict the energy dissipation pathways available for HBI after excitation by a photon, this restriction leaves fluorescence as the most readily available and common pathway for relaxation.  It was hypothesized by the [http://www.jaffreylab.org/Pages/default.aspx Jaffrey lab] that restricting motions of HBI using an RNA aptamer instead of proteinogenic amino acids could result in a similar fluorescence to GFP<cite>Jaffrey2011</cite>.  An aptamer is a small oligonucleotide which binds to a target molecule, they are identified from pools of random oligonucleotides through affinity selection<cite>Ellington1990</cite>. The group suspected that a modular, fluorescent RNA aptamer would facilitate RNA studies and RNA technologies, similar to the effect GFP has had on protein biochemistry and biotechnology.</div></td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>The natural fluorophore of GFP is formed through the auto-cyclization of three adjacent amino acids, Ser65, Tyr66, and Gly67, which react under oxidizing conditions to form hydroxybenzlidene imidazolinone (HBI).  [[File:HBI and GFP.png<ins class="diffchange diffchange-inline">|thumb</ins>|200px|right| A comparison of the GFP fluorophore after autocyclization and one of the HBI derivatives used by the Jaffrey Lab <cite>Jaffrey2011</cite>]] HBI itself is not significantly fluorescent, but becomes fluorescent when it is vibration restricted<cite>Meech2009</cite>.  Side chain contacts made with HBI in GFP prevent intramolecular motions, which restrict the energy dissipation pathways available for HBI after excitation by a photon, this restriction leaves fluorescence as the most readily available and common pathway for relaxation.  It was hypothesized by the [http://www.jaffreylab.org/Pages/default.aspx Jaffrey lab] that restricting motions of HBI using an RNA aptamer instead of proteinogenic amino acids could result in a similar fluorescence to GFP<cite>Jaffrey2011</cite>.  An aptamer is a small oligonucleotide which binds to a target molecule, they are identified from pools of random oligonucleotides through affinity selection<cite>Ellington1990</cite>. The group suspected that a modular, fluorescent RNA aptamer would facilitate RNA studies and RNA technologies, similar to the effect GFP has had on protein biochemistry and biotechnology.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>===Development and Physical Properties===</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>===Development and Physical Properties===</div></td></tr>
</table>Dst465https://www.synbiocyc.org/wiki/index.php?title=CH391L/S14/Spinach_RNA&diff=96417&oldid=prevDst465: /* References */2014-03-03T16:39:29Z<p><span dir="auto"><span class="autocomment">References</span></span></p>
<table class='diff diff-contentalign-left'>
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<td colspan='2' style="background-color: white; color:black; text-align: center;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black; text-align: center;">Revision as of 16:39, 3 March 2014</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 33:</td>
<td colspan="2" class="diff-lineno">Line 33:</td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>//EGFP paper</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>//EGFP paper</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>#Meech2009 pmid=19771336  </div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>#Meech2009 pmid=19771336  </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins style="font-weight: bold; text-decoration: none;">#Jaffrey2012 pmid=22403384 </ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins style="font-weight: bold; text-decoration: none;">//The spinach-SAM detection aptamer</ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div></biblio></div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div></biblio></div></td></tr>
</table>Dst465https://www.synbiocyc.org/wiki/index.php?title=CH391L/S14/Spinach_RNA&diff=96415&oldid=prevDst465: /* Uses */2014-03-03T16:38:56Z<p><span dir="auto"><span class="autocomment">Uses</span></span></p>
<table class='diff diff-contentalign-left'>
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<td colspan='2' style="background-color: white; color:black; text-align: center;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black; text-align: center;">Revision as of 16:38, 3 March 2014</td>
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<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>===Uses===</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>===Uses===</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>Spinach is useful as a modular appendage to RNA transcripts in the cell.  The addition of the 60 bp spinach aptamer to the end of coding and noncoding transcripts in cells has been shown to allow for monitoring of RNA localization in vivo.  The Jaffrey lab demonstrated this in the orginal spinach paper by appending spinach to the 3’ end of 5S ribosomal RNA<cite>Jaffrey2011</cite>.  By growing human cells with this modified gene in the presence of DFHBI, similar fluorescent patterns were seen as other experiments who had monitored 5S localization.  Additionally, when placed under stress, the cell responded by forming RNA granules colocalized with T-cell intracellular antigen-1-related protein, as was expected of the stress conditions.   </div></td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>Spinach is useful as a modular appendage to RNA transcripts in the cell.  The addition of the 60 bp spinach aptamer to the end of coding and noncoding transcripts in cells has been shown to allow for monitoring of RNA localization in vivo.  The Jaffrey lab demonstrated this in the orginal spinach paper by appending spinach to the 3’ end of 5S ribosomal RNA<cite>Jaffrey2011</cite>.  By growing human cells with this modified gene in the presence of DFHBI, similar fluorescent patterns were seen as other experiments who had monitored 5S localization.  Additionally, when placed under stress, the cell responded by forming RNA granules colocalized with T-cell intracellular antigen-1-related protein, as was expected of the stress conditions<ins class="diffchange diffchange-inline">.</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline">Jaffrey was also able to develop a spinach variant which is capable of detecting a second molecule, specifically S-Adenosyl-Methionine (SAM)<cite>Jaffrey2012</cite></ins>.  <ins class="diffchange diffchange-inline">The aptamer binds to both DFHBI and SAM, and only when bound to both small molecules does the complex fluoresce.  This technology could be expanded to a variety of small molecules, and used as a sensitive detection method.</ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>===Comparitive technologies===</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>===Comparitive technologies===</div></td></tr>
</table>Dst465https://www.synbiocyc.org/wiki/index.php?title=CH391L/S14/Spinach_RNA&diff=96413&oldid=prevDst465: /* Development and Physical Properties */2014-03-03T16:12:17Z<p><span dir="auto"><span class="autocomment">Development and Physical Properties</span></span></p>
<table class='diff diff-contentalign-left'>
<col class='diff-marker' />
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<td colspan='2' style="background-color: white; color:black; text-align: center;">Revision as of 16:12, 3 March 2014</td>
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<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>Individual sequences were screened for aptamers that accounted for the increased fluorescence observed, and the aptamer which gave the highest fluorescence was identified and named 13-2.  The Jaffrey lab then used truncations to determine a minimal aptamer [[File:Spinach derivatives.png|300px|right|A variety of aptamers against HBI derivatives results in a spectral range of fluoresence]]sequence, which resulted in an increase in quantum yield of fluorescence.  The final aptamer 13-2 is 60 nucleotides long, exhibited an emission peak of 529 nm, and an excitation peak of 398 nm.  Further selections allowed for tuning to a range of spectral properties, providing different color fluorescent molecules all using DMHBI as the substrate<cite>Jaffrey2011</cite>.   </div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>Individual sequences were screened for aptamers that accounted for the increased fluorescence observed, and the aptamer which gave the highest fluorescence was identified and named 13-2.  The Jaffrey lab then used truncations to determine a minimal aptamer [[File:Spinach derivatives.png|300px|right|A variety of aptamers against HBI derivatives results in a spectral range of fluoresence]]sequence, which resulted in an increase in quantum yield of fluorescence.  The final aptamer 13-2 is 60 nucleotides long, exhibited an emission peak of 529 nm, and an excitation peak of 398 nm.  Further selections allowed for tuning to a range of spectral properties, providing different color fluorescent molecules all using DMHBI as the substrate<cite>Jaffrey2011</cite>.   </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'>−</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>The quantum yield of GFP fluorescence was greatly improved by modification of the protein and screening, which lead to enhanced GFP, or EGFP.  The EGFP fluorophore is most often in the phenolate form  while wild type GFP is most commonly in the phonol form<cite>Tsien1998</cite><cite>Heim1995</cite>, this phenolate from is suspected to be the reason for EGFPs significant improvement in fluorescence.  Following that logic, the Jaffrey group set out to copy this by modifying the HBI derivative used in an attempt to obtain the phenolate form of HBI seen in EGFP.  The group swapped the methoxy-groups on the phenol ring to electron withdrawing fluorines, forming difluoro-HBI (DFHBI).  Selection for an aptamer against DFHBI resulted in an aptamer-DFHBI complex with significantly improved quantum yield over 13-2-DMHBI complex fluorescence.  The resulting complex shows 53% of the molar brightness of EGFP.  This aptamer, 24-2, was given the name “spinach”<cite>Jaffrey2011</cite>.</div></td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>The quantum yield of GFP fluorescence was greatly improved by modification of the protein and screening, which lead to enhanced GFP, or EGFP.  The EGFP fluorophore is most often in the phenolate form  while wild type GFP is most commonly in the phonol form<cite>Tsien1998</cite><cite>Heim1995</cite>, this phenolate <ins class="diffchange diffchange-inline">[[File:Phenolate DFHBI.png|100 px|right|The HBI derivative DFHBI in the phenolate form, which is more fluorescent]]</ins>from is suspected to be the reason for EGFPs significant improvement in fluorescence.  Following that logic, the Jaffrey group set out to copy this by modifying the HBI derivative used in an attempt to obtain the phenolate form of HBI seen in EGFP.  The group swapped the methoxy-groups on the phenol ring to electron withdrawing fluorines, forming difluoro-HBI (DFHBI).  Selection for an aptamer against DFHBI resulted in an aptamer-DFHBI complex with significantly improved quantum yield over 13-2-DMHBI complex fluorescence.  The resulting complex shows 53% of the molar brightness of EGFP.  This aptamer, 24-2, was given the name “spinach”<cite>Jaffrey2011</cite>.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>===Uses===</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>===Uses===</div></td></tr>
</table>Dst465https://www.synbiocyc.org/wiki/index.php?title=CH391L/S14/Spinach_RNA&diff=96411&oldid=prevDst465: /* Development and Physical Properties */2014-03-03T16:06:04Z<p><span dir="auto"><span class="autocomment">Development and Physical Properties</span></span></p>
<table class='diff diff-contentalign-left'>
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr style='vertical-align: top;'>
<td colspan='2' style="background-color: white; color:black; text-align: center;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black; text-align: center;">Revision as of 16:06, 3 March 2014</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 7:</td>
<td colspan="2" class="diff-lineno">Line 7:</td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>The group synthesized HBI derivatives and tested their hypothesis, first by exploring the fluorescence of HBI derivative ''in vivo,'' to ensure that HBI was not fluorescent under normal cellular conditions<cite>Jaffrey2011</cite>.  No significant fluorescence was detected from the HBI derivatives, and they proceeded by selecting for aptamers against the HBI derivative 3,5-dimethoxy-4-hydroxybenzylidene imidazolinone (DMHBI) using SELEX (systematic evolution of ligands by exponential enrichment).  After five rounds of selection, an increase in fluorescence was detected, and each subsequent round of selection saw an increase in fluorescence, up to the tenth round of selection.</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>The group synthesized HBI derivatives and tested their hypothesis, first by exploring the fluorescence of HBI derivative ''in vivo,'' to ensure that HBI was not fluorescent under normal cellular conditions<cite>Jaffrey2011</cite>.  No significant fluorescence was detected from the HBI derivatives, and they proceeded by selecting for aptamers against the HBI derivative 3,5-dimethoxy-4-hydroxybenzylidene imidazolinone (DMHBI) using SELEX (systematic evolution of ligands by exponential enrichment).  After five rounds of selection, an increase in fluorescence was detected, and each subsequent round of selection saw an increase in fluorescence, up to the tenth round of selection.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>    </div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>    </div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>Individual sequences were screened for aptamers that accounted for the increased fluorescence observed, and the aptamer which gave the highest fluorescence was identified and named 13-2.  The Jaffrey lab then used truncations to determine a minimal aptamer [[File:Spinach derivatives.png]]sequence, which resulted in an increase in quantum yield of fluorescence.  The final aptamer 13-2 is 60 nucleotides long, exhibited an emission peak of 529 nm, and an excitation peak of 398 nm.  Further selections allowed for tuning to a range of spectral properties, providing different color fluorescent molecules all using DMHBI as the substrate<cite>Jaffrey2011</cite>.   </div></td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>Individual sequences were screened for aptamers that accounted for the increased fluorescence observed, and the aptamer which gave the highest fluorescence was identified and named 13-2.  The Jaffrey lab then used truncations to determine a minimal aptamer [[File:Spinach derivatives.png<ins class="diffchange diffchange-inline">|300px|right|A variety of aptamers against HBI derivatives results in a spectral range of fluoresence</ins>]]sequence, which resulted in an increase in quantum yield of fluorescence.  The final aptamer 13-2 is 60 nucleotides long, exhibited an emission peak of 529 nm, and an excitation peak of 398 nm.  Further selections allowed for tuning to a range of spectral properties, providing different color fluorescent molecules all using DMHBI as the substrate<cite>Jaffrey2011</cite>.   </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>The quantum yield of GFP fluorescence was greatly improved by modification of the protein and screening, which lead to enhanced GFP, or EGFP.  The EGFP fluorophore is most often in the phenolate form  while wild type GFP is most commonly in the phonol form<cite>Tsien1998</cite><cite>Heim1995</cite>, this phenolate from is suspected to be the reason for EGFPs significant improvement in fluorescence.  Following that logic, the Jaffrey group set out to copy this by modifying the HBI derivative used in an attempt to obtain the phenolate form of HBI seen in EGFP.  The group swapped the methoxy-groups on the phenol ring to electron withdrawing fluorines, forming difluoro-HBI (DFHBI).  Selection for an aptamer against DFHBI resulted in an aptamer-DFHBI complex with significantly improved quantum yield over 13-2-DMHBI complex fluorescence.  The resulting complex shows 53% of the molar brightness of EGFP.  This aptamer, 24-2, was given the name “spinach”<cite>Jaffrey2011</cite>.</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>The quantum yield of GFP fluorescence was greatly improved by modification of the protein and screening, which lead to enhanced GFP, or EGFP.  The EGFP fluorophore is most often in the phenolate form  while wild type GFP is most commonly in the phonol form<cite>Tsien1998</cite><cite>Heim1995</cite>, this phenolate from is suspected to be the reason for EGFPs significant improvement in fluorescence.  Following that logic, the Jaffrey group set out to copy this by modifying the HBI derivative used in an attempt to obtain the phenolate form of HBI seen in EGFP.  The group swapped the methoxy-groups on the phenol ring to electron withdrawing fluorines, forming difluoro-HBI (DFHBI).  Selection for an aptamer against DFHBI resulted in an aptamer-DFHBI complex with significantly improved quantum yield over 13-2-DMHBI complex fluorescence.  The resulting complex shows 53% of the molar brightness of EGFP.  This aptamer, 24-2, was given the name “spinach”<cite>Jaffrey2011</cite>.</div></td></tr>
</table>Dst465https://www.synbiocyc.org/wiki/index.php?title=CH391L/S14/Spinach_RNA&diff=96410&oldid=prevDst465: /* Development and Physical Properties */2014-03-03T16:05:00Z<p><span dir="auto"><span class="autocomment">Development and Physical Properties</span></span></p>
<table class='diff diff-contentalign-left'>
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr style='vertical-align: top;'>
<td colspan='2' style="background-color: white; color:black; text-align: center;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black; text-align: center;">Revision as of 16:05, 3 March 2014</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 5:</td>
<td colspan="2" class="diff-lineno">Line 5:</td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>===Development and Physical Properties===</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>===Development and Physical Properties===</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>The group synthesized HBI derivatives and tested their hypothesis, first by exploring the fluorescence of HBI derivative ''in vivo,'' to ensure that HBI was not fluorescent under normal cellular conditions<cite>Jaffrey2011</cite>.  No significant fluorescence was detected <del class="diffchange diffchange-inline">in </del>the HBI derivatives, and they proceeded by selecting for aptamers against the HBI derivative 3,5-dimethoxy-4-hydroxybenzylidene imidazolinone (DMHBI) using SELEX (systematic evolution of ligands by exponential enrichment).  After five rounds of selection, an increase in fluorescence was detected, and each subsequent round of selection saw an increase in fluorescence, up to the tenth round of selection.</div></td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>The group synthesized HBI derivatives and tested their hypothesis, first by exploring the fluorescence of HBI derivative ''in vivo,'' to ensure that HBI was not fluorescent under normal cellular conditions<cite>Jaffrey2011</cite>.  No significant fluorescence was detected <ins class="diffchange diffchange-inline">from </ins>the HBI derivatives, and they proceeded by selecting for aptamers against the HBI derivative 3,5-dimethoxy-4-hydroxybenzylidene imidazolinone (DMHBI) using SELEX (systematic evolution of ligands by exponential enrichment).  After five rounds of selection, an increase in fluorescence was detected, and each subsequent round of selection saw an increase in fluorescence, up to the tenth round of selection.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>    </div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>    </div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>Individual sequences were screened for aptamers that accounted for the increased fluorescence observed, and the aptamer which gave the highest fluorescence was identified and named 13-2.  The Jaffrey lab then used truncations to determine a minimal aptamer sequence, which resulted in an increase in quantum yield of fluorescence.  The final aptamer 13-2 is 60 nucleotides long, exhibited an emission peak of 529 nm, and an excitation peak of 398 nm.  Further selections allowed for tuning to a range of spectral properties, providing different color fluorescent molecules all using DMHBI as the substrate<cite>Jaffrey2011</cite>.   </div></td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>Individual sequences were screened for aptamers that accounted for the increased fluorescence observed, and the aptamer which gave the highest fluorescence was identified and named 13-2.  The Jaffrey lab then used truncations to determine a minimal aptamer <ins class="diffchange diffchange-inline">[[File:Spinach derivatives.png]]</ins>sequence, which resulted in an increase in quantum yield of fluorescence.  The final aptamer 13-2 is 60 nucleotides long, exhibited an emission peak of 529 nm, and an excitation peak of 398 nm.  Further selections allowed for tuning to a range of spectral properties, providing different color fluorescent molecules all using DMHBI as the substrate<cite>Jaffrey2011</cite>.   </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>The quantum yield of GFP fluorescence was greatly improved by modification of the protein and screening, which lead to enhanced GFP, or EGFP.  The EGFP fluorophore is most often in the phenolate form  while wild type GFP is most commonly in the phonol form<cite>Tsien1998</cite><cite>Heim1995</cite>, this phenolate from is suspected to be the reason for EGFPs significant improvement in fluorescence.  Following that logic, the Jaffrey group set out to copy this by modifying the HBI derivative used in an attempt to obtain the phenolate form of HBI seen in EGFP.  The group swapped the methoxy-groups on the phenol ring to electron withdrawing fluorines, forming difluoro-HBI (DFHBI).  Selection for an aptamer against DFHBI resulted in an aptamer-DFHBI complex with significantly improved quantum yield over 13-2-DMHBI complex fluorescence.  The resulting complex shows 53% of the molar brightness of EGFP.  This aptamer, 24-2, was given the name “spinach”<cite>Jaffrey2011</cite>.</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>The quantum yield of GFP fluorescence was greatly improved by modification of the protein and screening, which lead to enhanced GFP, or EGFP.  The EGFP fluorophore is most often in the phenolate form  while wild type GFP is most commonly in the phonol form<cite>Tsien1998</cite><cite>Heim1995</cite>, this phenolate from is suspected to be the reason for EGFPs significant improvement in fluorescence.  Following that logic, the Jaffrey group set out to copy this by modifying the HBI derivative used in an attempt to obtain the phenolate form of HBI seen in EGFP.  The group swapped the methoxy-groups on the phenol ring to electron withdrawing fluorines, forming difluoro-HBI (DFHBI).  Selection for an aptamer against DFHBI resulted in an aptamer-DFHBI complex with significantly improved quantum yield over 13-2-DMHBI complex fluorescence.  The resulting complex shows 53% of the molar brightness of EGFP.  This aptamer, 24-2, was given the name “spinach”<cite>Jaffrey2011</cite>.</div></td></tr>
</table>Dst465https://www.synbiocyc.org/wiki/index.php?title=CH391L/S14/Spinach_RNA&diff=96408&oldid=prevDst465: /* Theory */2014-03-03T15:59:50Z<p><span dir="auto"><span class="autocomment">Theory</span></span></p>
<table class='diff diff-contentalign-left'>
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr style='vertical-align: top;'>
<td colspan='2' style="background-color: white; color:black; text-align: center;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black; text-align: center;">Revision as of 15:59, 3 March 2014</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 2:</td>
<td colspan="2" class="diff-lineno">Line 2:</td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>===Theory===</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>===Theory===</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>The natural fluorophore of GFP is formed through the auto-cyclization of three adjacent amino acids, Ser65, Tyr66, and Gly67, which react under oxidizing conditions to form hydroxybenzlidene imidazolinone (HBI).  [[File:HBI and GFP.png|200px|right|A comparison of the GFP fluorophore after autocyclization and one of the HBI derivatives used by the Jaffrey Lab <cite>Jaffrey2011</cite>]] HBI itself is not significantly fluorescent, but becomes fluorescent when it is vibration restricted<cite>Meech2009</cite>.  Side chain contacts made with HBI in GFP prevent intramolecular motions, which restrict the energy dissipation pathways available for HBI after excitation by a photon, this restriction leaves fluorescence as the most readily available and common pathway for relaxation.  It was hypothesized by the [http://www.jaffreylab.org/Pages/default.aspx Jaffrey lab] that restricting motions of HBI using an RNA aptamer instead of proteinogenic amino acids could result in a similar fluorescence to GFP<cite>Jaffrey2011</cite>.  An aptamer is a small oligonucleotide which binds to a target molecule, they are identified from pools of random oligonucleotides through affinity selection<cite>Ellington1990</cite>. The group suspected that a modular, fluorescent RNA aptamer would facilitate RNA studies and RNA technologies, similar to the effect GFP has had on protein biochemistry and biotechnology.</div></td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>The natural fluorophore of GFP is formed through the auto-cyclization of three adjacent amino acids, Ser65, Tyr66, and Gly67, which react under oxidizing conditions to form hydroxybenzlidene imidazolinone (HBI).  [[File:HBI and GFP.png|200px|right| A comparison of the GFP fluorophore after autocyclization and one of the HBI derivatives used by the Jaffrey Lab <cite>Jaffrey2011</cite>]] HBI itself is not significantly fluorescent, but becomes fluorescent when it is vibration restricted<cite>Meech2009</cite>.  Side chain contacts made with HBI in GFP prevent intramolecular motions, which restrict the energy dissipation pathways available for HBI after excitation by a photon, this restriction leaves fluorescence as the most readily available and common pathway for relaxation.  It was hypothesized by the [http://www.jaffreylab.org/Pages/default.aspx Jaffrey lab] that restricting motions of HBI using an RNA aptamer instead of proteinogenic amino acids could result in a similar fluorescence to GFP<cite>Jaffrey2011</cite>.  An aptamer is a small oligonucleotide which binds to a target molecule, they are identified from pools of random oligonucleotides through affinity selection<cite>Ellington1990</cite>. The group suspected that a modular, fluorescent RNA aptamer would facilitate RNA studies and RNA technologies, similar to the effect GFP has had on protein biochemistry and biotechnology.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>===Development and Physical Properties===</div></td><td class='diff-marker'> </td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>===Development and Physical Properties===</div></td></tr>
</table>Dst465