BE/CS 196a Lab 3: Design and analysis of

a smiley-shaped DNA origami

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1. DNA origami design (120 min)

Use Cadnano (tutorial videos) to design a smiley-shaped DNA nanostructure using the scaf-
folded DNA origami technique. Note that you do NOT need to install Autodesk Maya for
using Cadnano, and it is typically a lot easier to use Cadnano with a mouse. If you run into
OS compatibility issues, install the old version of Cadnano that works with Adobe Air.

Figure 1: Example block diagram and folding path of a smiley-shaped DNA origami.

Step 1 (20 min): calculate a scaffold path. To fold a smiley-shaped DNA origami us-
ing the full length (7,249 nt) of single-stranded viral DNA M13mp18 as scaffold, how
would you decide the number of bases on the longest double helix in the smiley shape
and the total number of rows of helices? The length of each base pair in a double helix
is 0.34 nm, and the height is 2 nm. We will use 1.5 turns standard spacing of staple
crossovers, which results in a 1 nm gap between two adjacent helices.

Hint: Let us first consider a disk, and calculate the area of a disk that can be folded
from the scaffold based on the total number of base pairs and the area of each base
pair, taking the inter-helix gap into consideration. The diameter of the disk can then
be calculated based on the area, and the number of base pairs on the longest double
helix can be calculated based on the diameter and the length of each base pair. The
number of helix rows can be calculated based on the diameter, the height of each base
pair and the inter-helix gap. Second, consider the smiley shape as a disk with three
holes, assuming that the holes are roughly 13% of the area of the disk.

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 Given the design constraint that there should be an integer number of turns between
two adjacent crossover locations on the scaffold if the crossovers are facing in the same
orientation, and an integer number plus half a turn if they are facing in opposite ori-
entations, could you better estimate the number of bases on the longest helix in the
smiley shape?

Step 2 (90 min): create the scaffold path.

a) With square lattice, click and select a column of helices in the left panel (side view
of the smiley shape), with the number of helix rows that you calculated. Slowly
drag your mouse from the top helix to the bottom to avoid skipping rows.
b) Click the helices again (also slowly from top to bottom) to create an initial raster
layout of the scaffold in the right panel (front view of the smiley shape).
c) Click the top right arrow in the right panel to add bases to all rows, according to
the number of bases on the longest helix that you calculated. Select all scaffold
crossovers on the right side and drag them to make all rows with the number of
bases on the longest helix.
d) Add the internal scaffold crossovers. When you click a row, all possible crossover
locations will be shown as corner brackets. Click a corner bracket to add a new
scaffold crossover.
e) Select and delete unconnected scaffold pieces in the eyes and mouth area.
f) Select and drag the edge scaffold crossovers to their correct locations.
g) Adjust all crossover locations to make the face, eyes and mouth look right.
h) Use the Break Tool to break the circular scaffold at the chin location. Place your
cursor near the break point, and the length of the scaffold will be shown at the
bottom left of the window. How many bases of M13mp18 did you end up using?
Is it close to 7,249?

Step 3 (5 min): create staples. Apply AutoStaple, and then AutoBreak with target
length 32, min length 20, max length 48 and min distance 8.

Step 4 (5 min): generate sequences. Use the Add Sequence Tool, click any part of the
scaffold and apply the M13mp18 sequence. Export the staple sequences as a csv file.

2. DNA origami analysis (60 min)

Use CanDo to analyze the smiley-shaped DNA origami.

Step 1 (20 min): generate images and movies predicting the 3D shape in solution and
the flexibility of the smiley-shaped DNA origami.

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 Step 2 (20 min): apply twist correction (delete 1 base in every three columns of staples,
resulting in 10.44 bases per turn of helix) to the DNA origami using the Skip Tool in
Cadnano.

Step 3 (20 min): compare the 3D shapes and flexibilities of the DNA origami before
and after twist correction.