OldAccordian

In this tutorial, we will use the .json file for a 53 basepair long two-helix bundle design where three insertions and deletions exist in neighboring helix. The corresponding .json file can be downloaded here.

Users may skip this step if they do not want an atomic model included with their results. Sequence information of the two-helix bundle is assigned using the “Add Sequence Tool” function in caDNAno. In this tutorial, we use the standard M13mp18 sequence. The sequence information of each staple strand is then exported in CSV format using the “export oligos as *.CSV” function in caDNAno. The corresponding .csv file can be downloaded here.

a. Click the red box (Submit a caDNAno file for analysis…) to expand the submission form

b. Enter user information (Name, Affiliation, and E-mail address)

c. DNA geometry

d. DNA mechanical properties

e. Model resolution

f. caDNAno (.json) file

g. Lattice type

h. Choose whether movies visualizing thermal fluctuations of the solution shape will be included with the results

i. Choose whether the atomic model of the solution shape will be included with the results

j. (Optional) caDNAno sequence (.csv) file

Pressing the submit button will automatically bring users to the result page.

Once the CanDo analysis is completed, users will receive results in ZIP files attached in separate emails sent to the email address they filled in the submission form.

a. Solution shape of DNA structures
Results in the BILD file format can be viewed using the UCSF Chimera.

b. Thermal fluctuations of solution shape
If users choose “Yes” in item “Would you like a movie included with your results?” in the submission form, users will receive a second email with a ZIP file containing movies (fluctuations_view1.avi, fluctuations_view2.avi, and fluctuations_view3.avi) showing thermal fluctuation in three orthogonal views. The structure in the movies is colored according to root-mean-square-thermal fluctuations.

c. Atomic model
If users choose to generate an atomic model in item “Would you like the atomic model included with your results?” in the submission form, users will receive a third email with a ZIP file containing the atomic model in PDB format as well as its images in three orthogonal views. The color of each strand in the atomic model is the same as in the caDNAno design (.json) file uploaded in the submission form. If users choose to visualize thermal fluctuations, movies in three orthogonal views are also included in the ZIP file.

CanDo assumes that all crossovers between neighboring helices in the design are located at their natural positions defined in the caDNAno at which torsional mismatch between neighboring helices is minimal. These natural crossover positions can be seen in the caDNAno design panel when a strand is clicked as shown in the figure below.

Currently, users must follow this crossover rule to design DNA origami structures for CanDo analysis. Below is an example two-helix bundle design that follows the crossover rule whose 3D solution shape is expected to be straight.

If a design does not follow the crossover rule, unexpected results may be obtained from the CanDo analysis. For example, in the modified two-helix design below, the right scaffold crossover is enforced at the position that is three basepairs left from its natural position as highlighted in the red box. Although there is torsional mismatch between basepairs connected by this crossover, expected to result in left-handed twist, CanDo predicts the straight solution shape because all crossovers are treated as natural ones.

To simulate the effect of this mismatch properly, the design should be modified using insertions or deletions with natural crossovers as below. Three deletions at each helix are added to the design while using natural crossovers only. CanDo predicts left-handed twist on the right half of the bundle correctly as expected.

Two more example design modifications are shown in the figure below where red dashed lines indicate natural crossover positions.

An algorithm to account for the mismatch due to unnatural crossovers is currently under development.

In wireframe structure design, crossovers between non-neighboring helices are used to connect substructures that are initially distant to each other but should be adjacent in the final relaxed configuration. CanDo defines these crossovers as distant crossovers that gradually shrink during the analysis until their length reduces to the distance between neighboring helices or the helix diameter. However, relative orientations of helices connected by distant crossovers remain unconstrained so that CanDo analysis is limited at present to on-lattice modeling of DNA origami structures. Thus, careful use of distance crossovers is advisable.

To illustrate this, let’s consider three test designs of four-helix bundle that have the same connectivity map. The first design consists of four neighboring helices with crossovers at the natural positions. Because all basepairs are aligned at crossovers, the deformed shape (right) is the same as the initial layout (left).

In the second design, the entire structure is divided into two sub-bundles (helices 0-1 and helices 2-3) separated vertically in the initial layout. Two sub-bundles are connected by distant crossovers between helix 1 and helix 2 at the same axial positions used in the first design so that basepairs connected by these distant crossovers are already torsionally aligned (highlighted with blue dashed lines). Hence the same deformed shape is obtained as in the first design.

In the last design, helices 2-3 are placed in an arbitrary position. Basepairs connected by distant crossovers between helix 1 and helix 2 are not torsionally aligned. In addition, all crossovers between helix 2 and helix 3 violate the natural crossover rule. In this case, the deformed shape predicted by CanDo will be different from that of the first and second designs.

We are currently developing a computational model to allow for more general crossovers, enabling lattice-free DNA origami design. Stay tuned!