Biomod/2011/TUM/TNT/Project/altStructure
<html> <meta http-equiv="Content-Type" content="text/html; charset=utf-8" />
<script type="text/javascript" src="http://ajax.googleapis.com/ajax/libs/jquery/1.4.2/jquery.min.js"></script> <script type="text/javascript"> $(document).ready(function(){ $('#lside1').mouseover(function() { $('#lside1').stop().animate({backgroundPosition: '0px 0px'}, 1000); }); $('#lside1').mouseleave(function() { $('#lside1').stop().animate({backgroundPosition: '-250px 0px'}, 1000); }); $('#lside2').mouseover(function() { $('#lside2').stop().animate({backgroundPosition: '0px 0px'}, 1000); }); $('#lside2').mouseleave(function() { $('#lside2').stop().animate({backgroundPosition: '-250px 0px'}, 1000); }); $('#lside3').mouseover(function() { $('#lside3').stop().animate({backgroundPosition: '0px 0px'}, 1000); }); $('#lside3').mouseleave(function() { $('#lside3').stop().animate({backgroundPosition: '-250px 0px'}, 1000); }); $('#lside4').mouseover(function() { $('#lside4').stop().animate({backgroundPosition: '0px 0px'}, 1000); }); $('#lside4').mouseleave(function() { $('#lside4').stop().animate({backgroundPosition: '-250px 0px'}, 1000); }); $('#lside5').mouseover(function() { $('#lside5').stop().animate({backgroundPosition: '0px 0px'}, 1000); }); $('#lside5').mouseleave(function() { $('#lside5').stop().animate({backgroundPosition: '-250px 0px'}, 1000); }); }); </script>
<style type="text/css">
#side1 {
width: 250px;
height: 100px;
position: relative;
top: 0px;
left: 20px;}
#lside1 {
width: 250px;
height: 100px;
background: url(http://openwetware.org/images/e/ee/1project.png) -250px 0px no-repeat;
position: absolute;
top: 0px;
left: 0px;}
#side2 {
width: 250px;
height: 100px;
position: relative;
top: 0px;
left: 20px;}
#lside2 {
width: 250px;
height: 100px;
background: url(http://openwetware.org/images/6/68/2results.png) -250px 0px no-repeat;
position: absolute;
top: 0px;
left: 0px;}
#side3 {
width: 250px;
height: 100px;
position: relative;
top: 0px;
left: 20px;}
#lside3 {
width: 250px;
height: 100px;
background: url(http://openwetware.org/images/f/f5/3methods.png) -250px 0px no-repeat;
position: absolute;
top: 0px;
left: 0px;}
#side4 {
width: 250px;
height: 100px;
position: relative;
top: 0px;
left: 20px;}
#lside4 {
width: 250px;
height: 100px;
background: url(http://openwetware.org/images/8/8f/4team.png) -250px 0px no-repeat;
position: absolute;
top: 0px;
left: 0px;}
#side5 {
width: 250px;
height: 100px;
background: url(http://openwetware.org/images/f/f5/3methods.png) -250px 0px no-repeat;
position: relative;
top: 0px;
left: 20px;}
#lside5 {
width: 250px;
height: 100px;
background: url(http://openwetware.org/images/1/11/5labbook.png) -250px 0px no-repeat;
position: absolute;
top: 0px;
left: 0px;}
</style> <style>
- column-one { display:none; width:0px;}
.container{background-color: #f5f5f5; margin-top:50px} .OWWNBcpCurrentDateFilled {display: none;}
- content { width: 0px; margin: 0 auto auto 0; padding: 1em 1em 1em 1em; align: center;}
- column-content {width: 0px; float: left; margin: 0 0 0 0;padding: 0;}
.firstHeading {display:none; width:0px;}
- globalWrapper{ width:1280px;}
- column-one {display:none; width:0px;background-color: #ffffff;}
- content{ margin: 0 0 0 0; padding: 1em 1em 1em 1em; position: center; width: 800px;background-color: #f5f5f5; }
.container{ width: 800px; margin: auto; background-color: #ffffff; text-align:justify; font-family: helvetica, arial, sans-serif; color:#555555; margin-top:25px; }
- bodyContent{ width: 1267px; align: center; background-color: #ffffff;}
- column-content{width: 1280px;background-color: #f5f5f5;}
.firstHeading { display:none;width:0px;background-color: #ffffff;}
- header{position: center; width: 800px;background-color: #ffffff;}
- footer{position: center;}
div.tright { border-bottom-width: 0em; border-left-width: 0px; border-right-width: 0px; border-top-width: 0em; clear: right; float: right;
}
<style type="text/css"> body {
font-family: helvetica, arial, sans-serif; color: black;
background:#f5f5f5;
}
ul#topnav {
margin: 0 0 0 0px ; padding: 0 0 0 0px; float: left; width: 800px; list-style: none; position: relative;
font-family: helvetica, arial, sans-serif;
font-size: 12px; background: #f5f5f5;
height: 5px;
} ul#topnav li { float: left; margin: 0; padding: 0; border-right: 1px solid #ffffff; /*--Divider for each parent level links--*/ } ul#topnav li a { padding: 11px 15px; display: block; color: #333333; text-decoration: none; } ul#topnav li:hover { background: #f5f5f5;} /*--Notice the hover color is on the list item itself, not on the link. This is so it can stay highlighted even when hovering over the subnav--*/
ul#topnav li span { float: left; padding: 15px 0; position: absolute; left: 0; top:30px; display: none; /*--Hide by default--*/ width: 800px; background: #cccccc; color: #333333; /*--Bottom right rounded corner--*/ -moz-border-radius-bottomright: 5px; -khtml-border-radius-bottomright: 5px; -webkit-border-bottom-right-radius: 5px; /*--Bottom left rounded corner--*/ -moz-border-radius-bottomleft: 5px; -khtml-border-radius-bottomleft: 5px; -webkit-border-bottom-left-radius: 5px; }
- abstractandlinks{
display:block; width:800px; background-color:#f5f5f5; text-align:justify;font-family: helvetica, arial, sans-serif; color:#555555; margin-top:0px; }
- innerBox
{ display: block; padding: 5px; }
- abstract{
font-family: helvetica, arial, sans-serif; width: 490px; margin-top: 20px; padding:0px 5px; float: left;background-color:#f5f5f5; ; }
- links{
font-family: helvetica, arial, sans-serif; width: 290px; margin-top: 20px; padding:0px 5px; float: left; background-color:#f5f5f5;; } .clear { clear:both; }
- struktur{
font-family: helvetica, arial, sans-serif; width: 790px; padding:0px 5px; float: left;background:#f5f5f5; }
- toctitle {align: left; text-align: left; }
- table.toc {background-color:"#5f5f5f";align: left; text-align: left;}
- toc #toctitle, .toc #toctitle, #toc .toctitle, .toc .toctitle {
text-align: left;
}
- toc, .toc, .mw-warning {
background-color: #f5f5f5; border-bottom-color: rgb(170, 170, 170); border-bottom-style: solid; border-bottom-width: 0px; border-left-color: rgb(170, 170, 170); border-left-style: solid; border-left-width: 0px; border-right-color: rgb(170, 170, 170); border-right-style: solid; border-right-width: 0px; border-top-color: rgb(170, 170, 170); border-top-style: solid; border-top-width: 0px; font-size: 105%; padding-bottom: 5px; padding-left: 5px; padding-right: 5px; padding-top: 5px; align: left; text-align: left; display: left
}
</style>
<head>
<script type="text/javascript"> var fullDates = new Array('08/11/2011','08/12/2011','08/16/2011','08/17/2011','08/23/2011','08/24/2011','08/25/2011','08/29/2011','08/30/2011','08/31/2011','09/01/2011','09/02/2011','09/06/2011','09/12/2011','09/13/2011','09/14/2011','09/15/2011','09/16/2011','09/19/2011','09/20/2011','09/21/2011','09/27/2011','09/28/2011','09/29/2011','09/30/2011','10/05/2011','10/06/2011','10/07/2011','10/10/2011','10/12/2011','10/13/2011','10/14/2011','10/17/2011','10/18/2011','10/19/2011','10/20/2011','10/21/2011','10/26/2011','10/27/2011','10/28/2011','10/30/2011');
$(document).ready(function() {
$("ul#topnav li").hover(function() { //Hover over event on list item $(this).css({ 'background' : '#cccccc'}); //Add background color and image on hovered list item $(this).find("span").show(); //Show the subnav
} ,
$("ul#topnav li").oneclick(function() { //Hover over event on list item $(this).css({ 'background' : '#cccccc'; 'display' : 'inline'}); //Add background color and image on hovered list item $(this).find("span").show(); //Show the subnav
} ,
function() { //on hover out...
$(this).css({ 'background' : 'none'}); //Ditch the background
//$(this).find("span").hide(); //Hide the subnav });
}); </script>
<script type="text/javascript" src="http://ajax.googleapis.com/ajax/libs/jquery/1.3/jquery.min.js"></script>
<title>
TUM NanU - Home
</title> </head>
<body background-color="#F0F0F0">
</body> </html>
Introduction
The structure is designed in order to measure the influence of DNA binders on the secondary structure of dsDNA. Therefor we decided to amplify the change in twist and pitch with an U-formed structure which is made of DNA it self by using 3D DNA origami and is capable of carrying FRET pairs on the arms for fluorescence measurements.
The principle of our structure is to use the change in the structure of dsDNA as a device and amplifier at the same moment. So we need a stiff base and connected to it two long deformable arms on which we can see in measurements the behavior of the DNA. Hence we want to use different devices for our measurements and controls (e.g. TEM, photospectrometer, fluorescence microscopy, FRET), we had to consider different aspects:
3D DNA origami
The all about of the Biomod competition is the design of bionano structures. The maybe best way of designing these thing is 3D DNA origamis structures. Let's see how this works: In principle we need to have a scaffold (in our case the genome of a bacteriophage called m13mp18) which is single stranded and several short oligonucleotides which are partially complementary to different places in the scaffold an staple it together. Therefor these little ssDNA pieces are called staples. Now let's look into the helical structure of dsDNA: it has a pitch of about 0.33 nm per base pair and 10.5 base pairs per turn. So the backbone of the helix turns 240 degrees within 7 base pairs. If you now place six helices in a honey comb lattice and connect them each 7 base pairs with each other, you have the most easy 3D origami structure in a honey comb lattice: a six helix bundle! With this basic principles you can derive many different and more complicated 3D structures, that have interesting properties. A good helper in design is caDNAno. For further information, visit (insert hendriks about caDNAno paper here). Now we know the principle and here we go...
Aspects of design
Distance between FRET pairs
The distance between the FRET pairs defines the distance of the arms of our structure in which it is sensitive for twist. In our case we are using ATTO 550 and ATTO 647N which have a Förster radius of about
[math]\displaystyle{ R_F = 6.5 nm }[/math].
Since we know the distance dependence of the FRET efficiency, we can derive a coarse distance of the two arms in order to have a mean FRET efficiency without DNA binders of about
[math]\displaystyle{ E_{FRET} = \frac{1}{2} }[/math]
[math]\displaystyle{ E_{FRET} = \frac{1}{1 + \left(\frac{r}{R_F} \right)^6} }[/math]
This means we need a distance of the FRET pairs of about [math]\displaystyle{ r = R_F }[/math].
Cross section of base
The only requirements for the cross section of the base is a very high stiffness and the ability to carry the two arms of our structure in a proper distance. Therefor we decided us for a 30 helix bundle in a honey comb lattice.
Cross section of arms
The design of the arms had to fulfill three main aspects:
- they are able to carry fluorophores at proper places for FRET measurements
- the cross section is asymmetric along the helical axis so the density projection in TEM pictures show us informations about twist of the arms
- in the end the arms should be a bit floppy so they can twist
We decided for a 10 helix bundle in a honey comb lattice, which should satisfy our requirements.
Position of FRET pairs
(on which helix, in which hight etc. )
Folding of the Structure
Purification (gel vs. filter purification)
labeling of FRET staples
immobilization
Inhalt:
Design, Warum ein U, Wo sind FRET Paare angebracht und warum und welches ist wofür gut (Twist oder Längenmessung)
Worauf muss man achten, damit das origami funktioniert, Ausbeuten vor/nach aufreinigung,
Probleme: Thermische Instabilität (laut cando)
(canDo, cadnano: oder bei Methoden??)
