02 DNA Nanostructures

This class was taught by William Shih from the Wyss Institute. He provided a presentation of his work on DNA origami and gave an introduction on his tool caDNAno, an open source software for designing DNA nanostructures.

DNA origami is the folding of DNA to create non-arbitrary two- and three-dimensional shapes at the nanoscale. The specificity of the interactions between complementary base pairs make DNA a useful construction material, through design of its base sequences. DNA is a well-understood material that is suitable for creating scaffolds that hold other molecules in place or to create structures all on its own.

Build a DNA origami structure

We begin the design of DNA origami using cadDNAno software. cadDNAno simplifies and enhances the process of designing three-dimensional DNA origami nanostructures. Through its user-friendly 2D and 3D interfaces it accelerates the creation of arbitrary designs. The embedded rules within cadDNAno paired with the finite element analysis performed by CanDo, provide relative certainty of the stability of the structures.

1. Build flattened Rothemund rectangle in cadDNAno

2. Generate staple strand sequences in caDNAno

3. Color staple strands to create a black-and-white pattern in caDNAno

JSON file output for the black-and-white pattern in cadDNAno:

4. Generate ASCII file black-and-white image

5. Generate list of wells to pipet to generate black-and-white pattern

By parsing the black-and-white ASCII pattern, we selected the appropriate empty and dumbbell staple DNA oligos from the respective 96-well plates. You can see the compiled pipeting instructions in Fig. 5. (The list is given in a 2D format, but it is essentially linear; you pipet everything and mix!

Generate caDNAno-format json file encoding 24 strand flat rectangle from a Python script

Experimental Protocol

Order from IDT the four 96-well plates needed to render 1-bit patterns ~$1500–$2000 for this set of strands on the 10 nmol scale (request 6 nmol total: 60 µL at 100 µM in water)

Assuming each pattern will consume 10 pmol per strand, this will be enough material to fold 600 patterns

Pipet strands (10 pmol each) to generate all patterns submitted by teams in the class

Fold patterns in a thermocycler in a volume of 100 µL per pattern

Check to see that the structures fold correctly by agarose-gel analysis of 15 µL aliquot

Find a partner (commercial or otherwise) willing to do AFM imaging of your samples

Submit your samples to that partner (only 1 µL required for imaging)