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Tutorial by Kasper Jahn and Ebbe S. Andersen

'''This tutorial takes you through the design process of DNA origami of an arbitrary shape inspired by the Rothemund, 2006 paper.'''

== Bitmap format ==

The way this program works is, that it takes the outline of the shape you give it and makes a DNA origami corresponding to the design pattern.

The program first finds out how the backbone and staple strands should be placed to make the DNA represent the figure you have given it. After finding out which geometry the strands need the sequence is added, depending on the size of the origami. This means that if you wish to exploit the entire M13 phage sequence you should give the input file a size (in pixels ) that would allow this.

Start with an image file and reduce it in size in e.g. Photoshop. The amount of black pixels should approximate the the size of the DNA strand to fold * 10 (approx. 70.000 for M13).

The DNA origami will slightly distort the image, since one turn is 3.6 nm and has a hight of 3 nm (Rothemund, 2006). We will make helixes in the x-direction, so the graphics file will be compressed in the y-direction. Therefore increase it the y-axis 1.2 fold.

We use the dolphin.bmp file as an example, that has 258 pixels from nose to back fin and 675 pixels from nose to tail. This corresponds to a size of 88 nm by 200 nm.

Save the graphics as a bitmap file in Windows File Format and 1 bit Depth. If you are using the "Paint" program in windows save the file as a monochrome.bmp. For some reason it is necessary to save the negative image with paint, so the figure is white where you want origami and black where you don't.

When you have a bitmap file saved in the right format, then you are ready to use SARSE. We will with this tutorial go through the process with a picture of a dolphin that is located in the tutorials-data folder. With whichever file you are using, it will later be necessary to type in the location (file path) of the file, so it would be an advantage to either write the file path down, or copy the path to paste in later.

Open the SARSE program. You should now see a pale blue background. Open a blank project, by choosing the file menu and select new. Go into the tutorial-data folder, and choose blank.txt. The picture that comes forth should look something like the picture below.


[[Image:Blank.png‎]]


You should now have a screen filled with pixels with dashes in all the pixels. We now want to import the bitmap file into the program. This is done by going into the Tools menu and choosing programs. You now have to select the DNA Origami program package, which contains all of the functions of the SARSE program. Go down and select the Import.bitmap.pl and select the Options button just to the right. This gives the opportunity to select the path from which to retrieve the bitmap file. Put a check in the small box above File Path, and enter the filepath of the bitmap file you wish to import, and press OK.


[[Image:Importbitmap.png‎]]


You will now see that not all of the pixels are dashes any more, but some of them are A's. This is not to confuse with the DNA base Adenine, but just a symbol to show that you wish the origami to fold here. Because the bitmap files are quite large you can only see a bit of them. if you are going through the tutorial with the dolphin.bmp file, you can only see the tip of the dolphin. To get a better overview of what is going on go into the View menu and choose Overview option. A new window will now open giving an overview of the entire figure. If you click on and point of the overview figure the image showing all of the pixels will zoom in to the area you clicked on. This is an excellent and clear way to navigate around the Origami instead of scrolling.


[[Image:Overview.png‎]]


You are able to change the symbols of the pixels on the screen. This is especially necessary if there are any sharp angles on the bitmap figure, because the origamis aren't too good at making tight sharp angles. The way to change the symbols in an area is to highlight the symbols that are to be changed by normal fashion. Then press alt+t, and a window will pop up where you can select which symbols you want to replace the old symbol with. So far we are only using the symbols "-" and "A" to indicate whether the pixel shall be part of the origami or not. When you have altered the pixels to satisfaction you are ready to get the backbone and the staple stands placed in the figure

== Automatic folding using helical twist ==

Now we want to fold a path through the shape, and the Origami-fold.pl program does this automatically for you. Choose the Tools menu and select programs. Here you choose the DNA-origami package. Select DNA-origami-fold.pl and click on the Options button. You have to stipulate 5 parameters for the fold.

[[Image:Origamifold4lille.png]]

B : Here you stipulate which row you want the origami to start in.

S : If you are making an origami with a seem, you can write which side of the origami you want the seam to be on (left or right), or if you want an origami without seams (no). A quick way of demonstrating which to choose is by looking at the way we made a dolphin with a seam. We split the dolphin in two and made an origami with each half. So for the left side of the figure you should choose right for the seam option, since the seam is on the right side, and vice-versa for the other half of the figure. By doing this you ensure that the dolphins can dock in to each other. To learn the program first, try making an origami without seams, since this is much simpler.

[[Image:Dolphins.png]]

H : Where to start folding from the top (left or right)

P : Which end of the DNA to start with (5p or 3p) corresponding to the 5´or 3´end.

O : The position of the first staple strand crossover. The parameter then becomes 10.5, because we want the first crossover to be between column 10 and 11. It is very important that the parameter is a "half" number.

The last paramter is sort of a reference frame for where it is possible to make crossovers, so that it is possible here at position 10.5, and at 10.5 plus any multiple of 32 along the y-axis, since the crossovers i one row are separated by 32 base pairs.

When you have selected the 3 parameters click the OK button.

The output file looks like this :

[[Image:Farverretning2.png]]

The path of the backbone is indicated with R, the staple strands with B and G (this will allow manual editing). The colour scale on the bases indicate the orientation of each base pair (red = up = 0 degrees; blue = down = 180 degrees). The helix twist of the backbone was calculated by adding the minor groove in the 5' to 3' direction (150 degrees), and adding the major groove in the 3' to 5' direction (210+180=390 degrees).

If you wish to see which strands are staple strands and which is the backbone you can see this by again accessing the DNA origami package (tools ==> programs ==> DNA origami ) and choosing color-symbols.pl. The view that comes is one where the backbone is Red, and the staple strands are Green and Blue. It is also again possible to see an overview of the entire origami by choosing View and selecting Overview. You should then see something like this :

[[Image:Redbluegreen.png]]

The fold path was found by the edges of the shape and adjusted for the helix twist of the backbone to point towards eachother. The crossovers are colored by the helix twist of the upper strand and thus every crossover should be blue and end on a red base on the lower strand. The program still has some problems at the edges, due to a bit looser demands on the discrete lengths of the individual row of the origami. Therefore it is necessary to correct the strands by editing some of the symbols.

== Editing the edges and adding features ==

In the middle of the origami, all the staple strands should be made correctly, but the automatically generated staple strands have some defects at the edges of the figure, and we therefore need to do manual editing. The editing is done by connecting or extending staple strands with B's og G's or deleting with "-" symbol. Select the positions in the editor and use the "Change symbol" icon in the side bar or the Edit menu and select Change symbol and choose G, B or -. A faster way is to select the positions which you wish to alter and press alt+t. When done changing symbols if you want the colors to correspond to the symbols, use the color-symbols.pl program (Tools menu ==> programs ==> DNA origami ==> color-symbols.pl ). You can see some examples of how this is done in the figures below.

[[Image:Beforeafter1.png]]

[[Image:Beforeafter3.png]]

[[Image:Beforeafter4.png]]

Now we want to add some extra decoration on the shape. To inhibit stacking of the helixes we add T-loops at the edges by inserting T's on the cross-over of staple strands on the edges. There are two different places where T´s must be added.

-Cross overs at edges

*Here the crossover B´s or G´s are removed and a T-linker is put in. It is still very important that there is base-pairing all the way to the end

-Staple strands that end at edges

*Here 2 T´s are added to the end of the staple strand

Both situations are illustrated on the figure below. The symbol we are using for T´s is a lowercase "u". As before the color-symbols.pl program can be used to change the colors, so they fit with the letters in the pixel.

[[Image:Tloop_before_after.png]]

On the dolphin shape we also want to add an eye. We click on the Overview window to navigate to the appropriate position and insert D's at the 3' end of a staple strand. We use helix twist to insert the D on a position that points either in to or out of the plane of the figure. The D will help us to insert a "dumbbell" DNA structure at this position, which is a bulky structure. In the figure below a uppercase "D" is inserted at the point where we want the dumbbell to be positioned.

[[Image:Beforeafterbumbbell.png‎]]

== Generating a 3-D view of the origami ==

This step is optional, but gives you the option of making a 3-D view of the origami. The first step is to download a pdb viewer. Pymol works on windows, linux and apple and can be downloaded at http://pymol.sourceforge.net. When you have pymol installed you can use the SARSE program to make a PDB file of the DNA origami that pymol can view.

After you have done all the final editing of the staple strands, select the tools menu, choose programs and select the DNA-origami package. Select the pdb-generator.pl program and fill in the parameter.

You must type the full file path for the sequence that you wish to use in the first line. The m13 sequence is located in the tutorial-data folder with the file name m13mp18.fasta. You can also use the browser to find the fasta file.


[[Image:Pdb.png]]


You then press OK and a pdb file is made. The location of the pdb file is in the folder of the project you are working on and the filename is pdbout.pdb.

Use pymol to open this file by opening pymol, selecting the file menu and then open. Browse until you find the pdb file. Underneath you can see pictures of the dolphin in pymol ( it is a slightly adjusted version, where we have put in a seam and an eye ). You can see that the DNA helices are closer to each other at the staple strand crossovers.


[[Image:Pymol1.png‎]]

== Adding sequence and printing oligos ==

Now we have finished the design of the DNA-origami and we want to find the primers to fold a particular sequence. Choose Tools menu and select programs. Again choose the DNA origami package. Put a check-mark at oligotrack.pl, and click on options. Mark both boxes that appear, and write the full path to the sequence-file (in fasta format) you wish to use. The m13 sequence is in the turorial-data folder, and has the file name m13mp18.fasta. You must also stipulate if the top strand in the fasta file runs forward (fw) or backwards (bw).


[[Image:Oligotrack.pl_small.png]]


Because of the linear design of the shape in this example we choose a M13 sequence that starts near the BsrI enzymatic cleavage site:

The track.pl program will insert the sequence in your folding path and find the sequence of the staple strands. The sequence of the staple strands is found by tracking from the 5' to the 3' end of the primers and written to a text file. Click ok and the result looks like this :


[[Image:Sequence.png‎]]


On the close-up of the origami the primers are annotated with a number, so it is possible to recognize them in the primer list. You can see these annotations on the figure above with a pink circle around them. The primer number corresponds to the pixel coordinates where the primer starts ( has a 5´end ).

The editor now shows the final sequence design with primer names written at the 5' end of each staple strand oligo. A primer list is written to a text file in the project directory, with the filename out2.txt and ready for ordering the oligoes. Please inspect the primer list in relation to the editor layout to accertain that the design is correct.


[[Image:Primerslille.png]]


These programs comes with no warranty, so make sure that your design is correct before ordering a large set of primers.

== References ==

* Andersen ES, Lind-Thomsen A, Knudsen B, Kristensen SE, Havgaard JH, Torarinsson E, Larsen N, Zwieb C, Sestoft P, Kjems J, Gorodkin J. 2007. Semiautomated improvement of RNA alignments. RNA 13:1850-1859.

* Rothemund PW. 2006. Folding DNA to create nanoscale shapes and patterns. Nature 440:297-302.