Biomod/2013/Aarhus/Results And Discussion/Peptide lock: Difference between revisions
| Line 4: | Line 4: | ||
=='''Introduction to peptide lock'''== | =='''Introduction to peptide lock'''== | ||
In order to keep the [[Biomod/2013/Aarhus/Results_And_Discussion/Origami#Origami_plate|plate]] and the [[Biomod/2013/Aarhus/Results_And_Discussion/Origami#Origami_dome|dome]] origami closely attached to each other a lock is needed. This lock should not only keep the construct assembled, but also open as response to a specific stimulus. In this case, this is the presence of the endoproteolytic enzyme matrix metallo protease 2 (MMP2) which is overexpressed by some types of metastasizing cancers.<cite>Mendes</cite> | In order to keep the [[Biomod/2013/Aarhus/Results_And_Discussion/Origami#Origami_plate|plate]] and the [[Biomod/2013/Aarhus/Results_And_Discussion/Origami#Origami_dome|dome]] origami closely attached to each other a lock is needed. This lock should not only keep the construct assembled, but also open as response to a specific stimulus. In this case, this is the presence of the endoproteolytic enzyme matrix metallo protease 2 (MMP2) which is overexpressed by some types of metastasizing cancers.<cite>Mendes</cite> | ||
==Matrix metalloprotease 2== | ==Matrix metalloprotease 2== | ||
Revision as of 04:42, 21 October 2013
<html> <style> /* ul.menu li.</html>Results_And_Discussion/Peptide_lock<html> a {
color: cyan;
}
- /
- toc {
display: none; }
- mytoc {
background: none; width: 200px; }
.toc { border: 0px solid; }
- toc ul ul,.toc ul ul {
margin: 0 0 0 1em; }
table.toc { background-color: #f0f4f4; }
- mytoc a,#mytoc a:visited {
font-size: normal; color: #222222; }
- mytoc a:hover {
font-color: #009ee0; /* text-decoration: underline; */ }
- wiki-toc {
width: 200px; margin-top: 6px; float: left; }
- wiki-body {
margin-left: 200px; padding-left: 12px; padding-right: 35px; }
- toc #toctitle,.toc #toctitle,#toc .toctitle,.toc .toctitle {
text-align: left; }
- toc h2,.toc h2 {
font-weight: normal; font-size: 17px; }
/*
- wiki-contents A {
color: #00aeef; }
- wiki-contents A:HOVER {
color: #00aeef; }
- /
- toctitle span {
display: none; }
/*
- wiki-body p,#wiki-body li,#wiki-body dd,div.thumbcaption {
font-size: medium; }
- /
/* required to avoid jumping */
- tocScrolWrapper {
/* left: 450px; */ position: absolute; /* margin-left: 35px; width: 280px; */ }
- tocScrol {
position: absolute; top: 0; /* just used to show how to include the margin in the effect */ /*margin-top: 20px; */ /* border-top: 1px solid purple; */ /*padding-top: 19px;*/ }
- tocScrol.fixed {
position: fixed; top: 0; }
- editPageTxt {
text-align: left; padding-left: 15px; }
- editPageTxt P {
clear: both; }
- toPageTop {
float: left; position: relative; top: 18px; left: 13px; color: #d13f31; } </style>
<script type="text/javascript"> $(document).ready(function() { var parentTable = $("#toc").parent(); $('#mytoc').append($("#toc").first());
$('#mytoc').find("#toc").attr("id", ""); parentTable.closest('table').remove(); });
$(document).ready( function() { var top = $('#tocScrol').offset().top - parseFloat($('#tocScrol').css('marginTop').replace( /auto/, 0)); var nav = $('#tocScrol'); var max = $('#indexing').offset().top - nav.height();
$(window).scroll(function(event) { // what the y position of the scroll is var y = $(this).scrollTop();
if (y > top) { // && signs are html decoded thus this construction if (y >= max) { nav.removeClass('fixed'); nav.css({ position : 'absolute', top : max - top }); } else { nav.addClass('fixed'); nav.removeAttr('style'); } } else { nav.removeClass('fixed'); nav.removeAttr('style'); } }); }); </script> <html> <html> <div id="wiki-contents"> <div id="tocScrolWrapper"> <div id="tocScrol"> <div id="wiki-toc"> <a id="toPageTop" href="#">▲</a> <table id="mytoc" class="toc" summary="Contents"> </table> <div id="editPageTxt"> <p> [<a href="http://openwetware.org/index.php?title=Biomod/2013/Aarhus/</html>Results_And_Discussion/Peptide_lock<html>&action=edit">edit this page</a>] </p> </div> </div> </div> </div> <div id="wiki-body"> </html>
Introduction to peptide lock
In order to keep the plate and the dome origami closely attached to each other a lock is needed. This lock should not only keep the construct assembled, but also open as response to a specific stimulus. In this case, this is the presence of the endoproteolytic enzyme matrix metallo protease 2 (MMP2) which is overexpressed by some types of metastasizing cancers.[1]
Matrix metalloprotease 2
MMP2, also known as gelatinase A, is an zinc-dependant 72 kDa endoprotease. {ref: D. E. Kleiner and W. G. Stetler-Stevenson. Matrix metalloproteinases and metastasis. Cancer. Chemother. Pharmacol.,, 43, 42-51, 1999. : 10.1007/s002800051097 } It is known to participate in the degradation of the extracellular matrix (ECM), which has countless biological consequences. {ref: R. Visse and H. Nagase. Matrix metalloproteinases and tissue inhibitors of metalloproteinases: Structure, function, and biochemistry. Circ. Res., 92, 827-839, 2003. DOI: 10.1161/01.RES.0000070112.80711.3D } Oncological studies have shown that some cancer types e.g. breast cancer, show increased MMP2 expression at the cell surface of metastases. {ref: O. Mendes, H.-T. Kim, G. Lungu, and G. Stoica. Mmp2 role in breast cancer brain metastasis development and its regulation by timp2 and erk1/2. Clin. Exp. Metastasis, 24, 341-351, 2007. DOI: 10.1007/s10585-007-9071-0 } As the enzyme degrades the ECM, it is easier for malignant cells to invade noncancerous tissue, and as ECM degrades, more space for the metastases is liberated. {ref: D. E. Kleiner and W. G. Stetler-Stevenson. Matrix metalloproteinases and metastasis. Cancer. Chemother. Pharmacol.,, 43, 42-51, 1999. : 10.1007/s002800051097 }
The lock was designed to contain the recognition and cleaving sequence of the enzyme {R. Visse and H. Nagase. Matrix metalloproteinases and tissue inhibitors of metalloproteinases: Structure, function, and biochemistry. Circ. Res., 92, 827-839, 2003. DOI: 10.1074/jbc.M109469200} , thus unlocking the construct in the vicinity of the cancer cell and exposing its functionalized interior. The protease is shown on figure XX {ref: E. Morgunova, A. Tuuttila, U. Bergmann, and K. Tryggvason. Structural insight into the complex formation of latent matrix metalloproteinase 2 with tissue inhibitor of metalloproteinase 2. Proc. Natl. Acad. Sci. USA, 99, 7414-7419, 2002. DOI: 10.1073/pnas.102185399}
The peptide sequence attempted incorporated was H-Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln-OH. {ref: E. I. Chen, S. J. Kridel, E. W. Howard, W. Li, A. Godzik, and J. W. Smith. A unique substrate recognition pro_le for matrix metalloproteinase-2. J. Biol. Chem., 277, 4485-4491, 2002. DOI: 10.1074/jbc.M109469200} The sequence was chosen based on its chemical simplicity, which accounts to a lesser chance of unwanted side reactions in the further processing of the molecule. The two approaches for the synthesis of the lock are elaborated in the following sections.
Synthetic methodology
The peptide
The peptides used for incorporation of the sequence into DNA is shown on figure XX. The glutamine was converted into its methyl ester derivative and the C-terminal was glycinol in the gradual build-up approach. For the fragment conjugation approach the native sequence was used and C-terminal was glycine. The Fmoc-protected methyl ester derivative was succesfully succesfully(REF TIL LITETARUTUERU).
For N-terminal linker of the peptide, pent-4-ynoic acid, has been covalently coupled onto the peptide using standard peptide coupling reagents. For the C-terminal linker either glycinol or glycin is used. These derive from the solid support used in the solid phase peptide synthesis.
The DNA sequence
The DNA sequence that was used for the lock is shown below. The length and composition of the strand was based on design by NUPACK {ref: J. N. Zadeh, B. R. Wolfe, and N. A. Pierce. Nucleic acid sequence design via efficient ensemble defect optimization. J. Comput. Chem., 32, 439-452, 2011. DOI: 10.1002/jcc.21633} to have the least amount of intramolecular interactions. The oligo length was selected based on the design of the plate and dome DNA origami. Furthermore the design allowed for the structure to "breath", as one end of the lock act as a zipper, matching 5' with 5'. The other end was antiparallel, thus forming a rigid structure. (ANE SKAL SKRIVE NOGET A FDET HER MULIGVIS HUHEUEH)
5'-GAACTCTCAGCTTATCATGC-3'
5'-GCTAAAGACCTATATCTGCGGTGAA-3'
Gradual buildup
The first approach in the synthesis of the lock was to utilize the fully automated oligonucleotide synthesis, in which a gradual build-up of DNA is achieved. According to literature, {ref:T. Moriguchi, T. Yanagi, M. Kunimori, T. Wada, and M. Sekine. Synthesis and properties of aminoacylamido-amp chemical optimization for the construction of an n-acyl phosphoramidate linkage. J. Org. Chem., 65, 8229-8238, 2000. DOI: {10.1021/jo0008338} {ref: A. Grandas, J. Robles, and E. Pedroso. Phosphitylation of primary carboxamides. synthesis of peptide-oligonucleotide conjugates with acylphosphoramidate linkages. Nucleosides Nucleotides Nucleic Acids, 14, 825-828, 1995. DOI: 10.1021/jo00120a032} {ref: J. Robles, E. Pedroso, and A. Grandas. Peptide-oligonucleotide hybrids with n-acylphosphoramidate linkages. J. Org. Chem.,, 60(15):4856{4861, 1995. DOI: 10.1021/jo00120a032} the glutamine moity exhibits nucleophilic features in the used standard phosphoramidite approach. To circumvent this problem, the amide was masked as a non-nucleophillic group , its methyl ester derivative. Once the peptide was coupled onto the DNA, a small organic molecule, 2-(2-(2-Azidoethoxy)ethoxy)ethanol, was coupled to the DNA peptide conjugate. The product of the click reaction would result in a free alcohol on which the oligo nucleotide synthesis could continue. The methyl ester derivative could after the synthesis has completed, be converted back to the glutamine residue thus resulting in the original sequence.
Fragment conjugation
The second approach was to conjugate DNA to the peptide sequence using standard peptide coupling conditions and click reaction. This approach does not require the glutamine residue to be masked, thus the native DNA sequence can be used. For this approach azide and amine modified DNA strands are needed. These were synthesized using standard conditions and reagents on the automated oligonucleotide synthesizer. Figure XX shows the setup for the approach.
Results and discussion
Gradual Buildup
The initial DNA strand was synthesized on the automated oligonucleotide synthesizer. {hyperlink MErMADE}. The approach in which the peptide was activated with phosphoramidite prior to the addition to the automated oligonucleotide synthesizer did not yield any coupling products. The only oobserved mass in MALDI-TOF analysis was of the truncated DNA strand. Therefore the approach of phosphoramidite activation of DNA was preferred.
Purification after peptid coupling
The oligo nucleotide was synthesized and activated prior to the addition of peptide. The coupling was attempted and the resulting mixture was cleaved off solid support, purified and analyzed. The analysis did not yield any DNA sequences according to LCMS, even though UV/VIS confirmed that the probe contained DNA as it showed absorbance in the 260 nm region. A PAGE analysis was performed and yet again no DNA was found.
It was suspected that the possible DNA-peptide conjugate would form micelle once cleaved into solution in the purification step. To verify whether or not aggregates are formed, Dynamic Light Scattering (DLS), measurements were performed. The DLS measurements are shown in table XX.
This concludes that micelles have formed once the DNA-peptide conjugate were cleaved into solution.
Purification after total synthesis
To circumvent the micelle formation in solution, the strategy was to clickthe small organic molecule onto the DNA-peptide conjugate and continue the synthesis
The free 5’ alcohol of the 25 mer was then in situ activated with phosphoramidite {hyperlink: phosphoramidite approach} prior to the addition of the peptide. After the hypothetical peptide coupling, the small organic molecule was attempted coupled onto the molecule. On the now free alchohol, continued oligonucleotide synthesis was attempted. The product was cleaved off solid support, purified and analyzed by MALDI-TOF {hyperlink MALDI-TOF} and LCMS {hyperlink LCMS}.
The mass of the DNA-peptide-DNA conjugate was not observed in either mass spectra. The observed mass by MALDI-TOF was approximately that of the DNA-peptide conjugate, using a linear positiv mode. The peak was broad, and therefore inconclusive.
Fragment conjugation
The second approach is to synthesize each fragment separately and conjugate them afterwards. The before mentioned 20 and 25- mer were synthesized on automated oligonucleotide synthesis with a 3’ and 5’ amine modification, respectively. This modification allowed the use of standard peptide coupling conditions for the conjugation of the C-terminal carboxillic acid and the amine modified DNA strand. The other DNA strand was afterwards modified with an small molecule with an azide, thus making the click coupling between the N-terminal alkyne and the DNA possible.
Both amine modified DNA strands and azide modified DNA strand was successfully synthesized.
Amide bond formation
As numerous attempts (LINK til experiemntals)in conjugating the amine modified DNA strand with the peptide with standard peptide coupling chemistry failed, the click reaction experiments were initialized.
Conjugation through click reaction
The click reaction between the azide modified DNA and the peptide successfully yielded the desired molecule, as confirmed by MALDI-TOF analysis. The yield was low, only 0.14%.
Template direction
To increase the local concentration of the reactive moieties a DNA template directed approach was attempted. The template was designed to be complimentary to both DNA strands of the lock. A fragment of multiple thymine bases inbetween the complimentary strand corresponds to the length of the peptide. The setup is seen on figure XX.
As the DNA-peptide conjugate was succesfull, the template mediated reaction was performed. Coupling with EDC/NHS and DM-TMM with and without template was attempted, and native and denaturated PAGE analysis was performed.
The DNA-peptide conjugate was not purified sufficiently, which is the reason multiple bands are present in the corresponding lane. The PAGE analysis (figure xx) shows that no additonal bands are formed in neither one of the condition, thus concluding that the desired molecule was not formed.
Conclusion
The DNA-peptide conjugate was succesfully optained by click reaction. The amide bond formation between the amine modified DNA strand and the DNA-peptide conjugate was unsuccesfull and no further experiments where initiated due to time limitations of the BIOMOD competition.
<html></div></div></html> <html> <head> <style>
- indexing {
/* float: left; position: center; */ background-color: #222; border-top: 2px solid #d13f31; color: #006e9c; margin: 0px; padding: 0px 0px 10px 0px; width: 100%; text-align: center; }
.footer-section { padding: 10px; display: table-cell; text-align: left; }
.footer-section-title { font-size: 20px; }
- footer-contents {
color: #006e9c; display: inline-table; }
.footer-section A { color: #006e9c; text-decoration: none; }
.footer-section A:HOVER { color: #00aeef; }
.footer-section ul { list-style-type: square; }
- sitemapTitle {
margin-top: 20px; font-size: 24px; }
- editFooter {
float: right; margin-top: -28px; margin-right: 5px; }
- editFooter A {
color: #006e9c; text-decoration: none; }
.cf:before,.cf:after { content: " "; /* 1 */ display: table; /* 2 */ }
.cf:after { clear: both; }
- bodyContent a[href^="mailto:"], .link-mailto {
background: url() no-repeat scroll right center transparent; padding-right: 0px; color: #006e9c;
}
</style> </head> <body> <div id="indexing"> <div id="sitemap"> <p id="sitemapTitle">SITEMAP | BIOMOD 2013 NANO CREATORS | Aarhus University</p> <div id="footer-contents"> <div class="footer-section"> <p class="footer-section-title">INTRODUCTION</p> <ul> <li><a href="/wiki/Biomod/2013/Aarhus">Home, abstract, animation and video</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Introduction">Introduction</a></li </ul> </div> <div class="footer-section"> <p class="footer-section-title">RESULTS AND DISCUSSION</p> <ul> <li><a href="/wiki/Biomod/2013/Aarhus/Results_And_Discussion/Origami">Origami</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Results_And_Discussion/Peptide_lock">Peptide lock</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Results_And_Discussion/Chemical_Modification">Chemical modification</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Results_And_Discussion/sisiRNA">sisiRNA</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Results_And_Discussion/System_In_Action">System in action</a></li> </ul> </div> <div class="footer-section"> <p class="footer-section-title">MATERIALS AND METHODS</p> <ul> <li><a href="/wiki/Biomod/2013/Aarhus/Materials_And_Methods/Origami">Origami</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Materials_And_Methods/Peptide_lock">Peptide lock</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Materials_And_Methods/Chemical_Modification">Chemical modification</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Materials_And_Methods/sisiRNA">sisiRNA</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Materials_And_Methods/System_In_Action">System in action</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Materials_And_Methods/Methods">Methods</a></li> </ul> </div> <div class="footer-section"> <p class="footer-section-title">SUPPLEMENTARY</p> <ul> <li><a href="/wiki/Biomod/2013/Aarhus/Supplementary/Team_And_Acknowledgments">Team and acknowledgments</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Supplementary/Optimizations">Optimizations</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Supplementary/Supplementary_Data">Supplementary data</a></li>
<li><a
href="/wiki/Biomod/2013/Aarhus/Supplementary/Supplementary_Informations">Supplementary informations</a> <li><a href="/wiki/Biomod/2013/Aarhus/Supplementary/References">References</a></li> </ul> </div> </div> <div> <p id="copyright">Copyright (C) 2013 | BIOMOD Team Nano Creators @ Aarhus University | Programming by: <a href="mailto:pvskaarup@gmail.com?Subject=BIOMOD 2013:">Peter Vium Skaarup</a>.</p> </div> </div>
<!-- Sponsers --> <div> <img alt="Sigma - Aldrich" src="http://openwetware.org/images/3/39/Sigmaaldrich-logo%28transparant%29.png" width="300px" height="154px"> <img alt="VWR International" src="http://openwetware.org/images/2/28/Vwr_logo.png" width="300px" height="61px"> <img alt="Promega" src="http://openwetware.org/images/7/72/Promega.png" width="175px" height="105px" style="padding-right: 5px; padding-left: 5px;"> <img alt="kem-en-tec" src="http://openwetware.org/images/3/3a/Kementec.png" width="130px" height="129px"> <img alt="Centre For Dna Nanotechnology" src="http://openwetware.org/images/4/4f/CDNA_logo.png" width="420px" height="90px"> <img alt="Dansk Tennis Fond" src="http://openwetware.org/images/9/9a/Dansk_tennis.png" width="250px" height="53px"> </div> <div id="editFooter"> [<a href="http://openwetware.org/index.php?title=Template:Biomod/2013/Aarhus/Nano_Creators/footer&action=edit">edit sitemap</a>] </div> </div> </body> </html>
