DNA OrigamiMaking an unlockable box out ofMCB Journal ClubPresented by Owen Woody

Wednesday July 22nd12:30-1:30pm Deans Conference Room

Todays Agenda An introduction to the DNA origami methodology Scaffolds, staples, bridges, pixels

A brief history of DNA origami Three hole discs and dolphins

The DNA box itself Proving the box is a box Lock and key mechanism

How small are these things?

That scale bar says 50 micrometers. This house dust mite is around 400 micrometers by 300 micrometers. Most of the scale bars well see today are in nanometers, a thousandth of a micrometer.

DNA origami methodology Largely based on the milestone paper: Rothemund, P. (2006) Folding DNA to create nanoscale shapes and patterns Nature, Vol 440. The following tutorial on the technique was largely borrowed from this paper.

Usual scaffold is M13mp18 viral genome, 7249-nt single stranded. This design requires a 900 nt scaffold.

Yellow dot marks a spot where the seam can be bridged by a staple.

Creating pixelsPixels are created by modifying staple strands. Staples can be given biotin tags, etc. In this paper, a dumbbell hairpin is used, causing the flagged staples to have slightly more thickness under the AFM tip.

Aside: how an AFM works (in extremely simple terms)

H, I show stacking along blunt ends

A field was born This paper demonstrated that DNA origami could cut a lot of corners No purification of staple strands, no precise stoichiometry between staples and scaffold Avoided staples binding staples Did not worry about secondary structures

An upper limit on sophistication The process does require uniqueness of sequence content (staples all cover specific unique ranges) Thus, theres an upper limit on the scale of the product that can be produced in a single run

Next paper: Dolphin software The next paper Ill cover in some detail comes from the same lab that went on to produce the DNA box. Andersen et al. (2008) DNA Origami Design of Dolphin-Shaped Structures with Flexible Tails ACS Nano vol. 2 No.6

Importantly, they created software

Dolphin design One goal of this study was to gauge whether the flexibility of the product could be controlled by restricting stapling across seams

Specifically, the tail region of the dolphin was designed to be flexible.

Flexible Tails

Interestingly, dolphin 1 in figure B (top right) has a very blurry tail this dolphin is oriented perpendicular to the direction of AFM scanning, suggesting the tail region was knocked around by the probe.

Controlling flexibility with staples

Making Dolphin Logos

On to the box3D origami structures, a natural next step, followed shortly thereafter. In fact, the paper Im presenting today had a couple rivals this image is from a paper submitted by a Japanese group that same year. Perhaps the lock/key mechanism was what gave Andersens group the edge.Kuzuya and Komiyama (2009)

DNA Box manufacturing steps

The depth was less than expected in the absence of cargo, the hollow box seemed prone to collapse under the force of the probe from the AFM. See supplement for theoretical models of collapsed structures.

Further attempts to prove its a box

Cryo-electron microscopy not my field of expertise, but thick lines apparently reflect dense faces.

Lock and Key mechanismLock uses strand displacement. In closed position, FRET causes only one emission wavelength to be detectable In the open orientation, FRET effects are diminished and both signals are present.

Possible Cargo Large enough to contain a ribosome (20nm diameter) Poliovirus State the box can be opened and then resealed I wonder how youd encourage cargo into the box?

Take-home points DNA origami is a relatively low-cost means of producing nano-scale structures The field is no longer niche several labs in the world now involved. Software is available for designing basic DNA origami templates from provided bitmaps (make your own likeness out of DNA!)