The overall goal of this procedure is to design a robotic device from DNA origami using the CAD nano software. This is accomplished by first installing CAD nano and using it to outline the general shape of the robot. The second step is to design the path of the scaffold strand.
Next, the staples are inserted and edited. The final step is setting control features, including payload, attachment sites and gates. Ultimately, a list of DNA parts is generated for ordering prior to assembly.
The main advantage of this technique is that it simplifies and standardizes the design of DNA origami objects for diverse applications. Demonstrating the procedure will be Alad Bei, a postdoc, working on the dynamics of DNA origami robots in my laboratory. To begin this procedure, download and install CAD nano two and Autodesk Maya 2012.
According to the instructions on http slash slash cad nano. org, the design interface of CAD nano within Maya includes three panels. The top panel is the lattice view where the shape is initially outlined.
This panel enables double helix level actions and provides a section view of the shape. The bottom panel is the editing panel, enabling single base level actions. The right panel is a Maya generated real-time 3D model of the shape.
Click the honeycomb icon zooming in, on and out of the lattice. In the top panel can be done by mouse. Scroll up and down respectively.
To begin, draw the section of the desired shape on the left panel. Each circle represents a double DN, a helix to choose the helixes, which build the shape, simply left click on their center. Continue helix by helix until the entire shape is outlined.
Alternatively, the shape can be drawn by pressing the left mouse button and continuously drawing the shapes outline. Observe the bottom panel. Each helix is represented by two rows of squares.
The rows are the two strands of the double helix. With each square representing a base, the orange vertical bar determines where editing actions take place along a helix. The base position along the grid appears as a number above the orange bar.
The helix framework's default length is 42 bases. The length can be extended by clicking one of the gray arrow icons at the top right corner of the editing panel and choosing the extension length. To plot the actual scaffold strand path throughout the shape, press the mouse button.
Start from the first helix and go continuously over all the helices following the same order they were initially selected. Draw a rectangle around all the leftmost edges of the scaffold path. Extend the scaffold path by dragging the selected edges as a group to the left side of the grid.
After repeating this process until the path is properly extended and identifying bridge icons as described in the written text, prepare to create crossovers to create crossovers. Left click the bridge icon of choice. A scaffold crossover will be generated, meaning the scaffold crosses at this point from helix to helix.
Repeat this process until the scaffold traverses all helixes and create a closed loop that spans the entire shape, leaving no regions that are isolated from the rest of this shape. The described robot opens in response to a defined biological input to expose its payload. Opening takes place in a shell like manner with two halves revolving around two axes.
The axes are formed by crossovers between helices 29 and 30 and 61 and zero, which are the only crossovers between those halves and are positioned only in or close to the left edge of the grid. The right edge will contain the gait strands. Erase the existing crossover between helices 29 and 30.
To erase the crossover, click the knee point in either strand. This leaves the nick in both strands where the crossover used to be. To seam the Nicks press shift and click each nick, create a new crossover between helices 29 and 30 as close as possible to the left edge of the grid.
Then create a new crossover between helices 61 and zero as close as possible to the left edge of the grid, define four loading sites facing towards the internal side of the robot. The loading sites will branch out of Helices 3 27, 34, and 58. For each site in the top panel, click the helix immediately adjacent to these helices that faces the internal side.
This will add the helixes to the grid in the bottom panel. Do not second click these lyes yet to add and edit staples. First click auto staple.
The software will automatically add staple sequences in various colors. Note that staples have been added to the 3D shape in the right panel. Staple colors are consistent for the bottom and right panels.
In addition, there is an indicator on the bottom left corner of the interface, which indicates a staple staples cannot be too long, too short or circular to automatically edit staples. To meet these criteria, click auto break, a dialogue box will open asking for user-defined parameters for this action, use the default parameters and click okay. The software will break the staples according to these parameters to the best of its ability.
Erase all the staple crossovers between helices 29 and 30 and 61 and zero to enable these helices to separate and enable the robot to open. Erasing staple crossovers will require some manual editing to correct staples that become too short or irrational as a result of this action. To do this properly, follow the instructions in the text protocol seam, the two staples on Helix 29 by pressing shift and clicking the nick between them.
Similarly seem the three staples on Strand 30 to a single staple staples can be manually extended or shortened by clicking an edge and dragging it as desired. Take care not to circularize any staple. Repeat this process for HELOCs zero and 61 and manually edit all the staples in each helix.
Locate staples that are drawn by a thick line, meaning they require further editing. Examine each one and correct as necessary. For example, staples that are too short can be erased or extended if possible.
Second, click the loading site helices in the top panel and extend the resulting scaffold strand fragments in the bottom panel by clicking an edge and dragging it as desired. Manually add staples to these scaffold fragments by placing the orange vertical bar at the desired position Along the scaffold, going over the guide helices on the left panel holding shift and clicking. This will add a staple precursor at each helix.
Extend the staple precursors to full length as well By clicking and dragging, locate the red bridge icons denoting a loud crossover points between the guide strand and the chassis. Choose the most convenient location to introduce a crossover and click the bridge icon. A convenient location requires minimal editing of existing staples in the chassis.
In the guide helix, delete the staple part that is not a part of the loading site and shorten the participating part to the desired length. The desired length should provide both specificity for loading different types of cargo and binding strength. Typically, an 18 mer tail should be fine.
Make sure the staple remains drawn by a thin line. Otherwise, edit until it is in the chassis. Edit the changed staples as necessary.
Erase the guide, leaving only the staple extension. Repeat these steps for all loading sites to design gait strands. First, locate the proper positions for gait strands.
These will be staples on helices 29, 30 61 and zero. For example, examine the 29 to 30 gait region. There are convenient staple strands, flanking helices 29 and 30 on the right side of the grid, which can be used as gate strands.
Note that they face opposite directions. Click the edge of one of the potential gait strands to extend it outside of the shape. If the edge lies over a scaffold crossover, it selection could be simplified by making sure only staple are selectable.
By clicking off scaf in the selectable toolbar on the top right side of the interface, extend both staples to form the gate strands. Edit the staples. If this extension requires it, repeat this for the gate strands of helices zero and 61.
To choose the scaffold sequence, click the seek tool. Place the cursor anywhere on the scaffold strand and click a dialogue box will open asking to choose the scaffold DNA source In the dialogue box, click M 13 MP 18. Note that the chosen DNA sequence has been added to the scaffold and staple strands in the bottom panel.
Finally, export the staple sequence as a spreadsheet by clicking export on the top toolbar and choosing a destination file name for the staple list. Click save. This video has demonstrated a step-by-step process for the design of A DNA origami nano robot.
The cross section of the shape was first outlined here. The outline was followed by automatic edition of scaffold strand fragments and completion of the entire scaffold path. Shown here is a general view of the bottom and right panels demonstrating how the 3D model changes in real time along with editing actions.
Staple strands were automatically added as can be seen in the blueprint after the auto staple action. In this image, the staple colors in the bottom panel and the right panel are consistent. The staple strands were then broken according to user-defined parameters as shown here in the blueprint after the auto brake action.
Next, the staple strands were manually edited to adapt the staples to the desired function of the device. The entire gap between Helices 29 and 30 show that no crossovers link the two in this blueprint. After manual editing shown here is a view of the loading site staples as seen in the bottom panel.
After removing the guide HELOCs, which are no longer necessary, this image shows extending of two staples, which are going to be used as GA strands from HELOCs 29 and 30. Note that the two strands face opposite directions, which is mandatory for the formation of the gait duplex. This is the TEM image of the final model designed in this procedure.
The sample is stained with urinal format and imaged in a GL transmission electron microscope exactly as described in previous reports by the authors. After watching this video, you should have a good understanding of how to design a robot from DNA origami for any application of your choice.
