Design

 

The Video Script

The starting point of our project is to reengineer DNA-protein complexes. What you’re looking at here is an atomic detail model of one of our designable co-crystals: it contains a short double stranded DNA duplex with two monomers of the protein.

To prevent confusion, we will use a simplified representation. Now, we’ve turned on the cartoon view and we can easily see the double helix being bound by two proteins. This protein-DNA complex will self-assemble into a co-crystal in which blocks line up at the DNA-DNA interfaces.

The DNA stacks end-to-end with a blunt end interaction. There are no sticky ends joining the DNA in the original co-crystals.

We can also represent DNA binding proteins using LEGO bricks. The white blocks are protein while the grey rods are DNA. These components can make a tight protein-DNA complex under the right conditions. A lattice structure is built with protein-protein contacts shown as stacking and DNA-DNA interactions shown as red linker pieces, allowing the crystal to grow in two dimensions.  However, since the crystal is held together using only weak interactions, it is mechanically fragile and very sensitive to its environment.

We’re going to further simplify the cartoon by showing you cylinder views of the DNA. This is a good representation because DNA is a pretty rigid molecule. Now, we’re showing you the two different proteins with different colors: one in yellow and one in green.

In order to expand the original crystal, our design consists of a DNA strut being inserted into the original DNA. The inserted new block is shown here as a dark green cylinder.

While in detail this is one way to accomplish the DNA expansion, it is easier to handle two pieces of DNA rather than three. Let’s zoom around and see how we will actually create only one breaking point in the DNA junction, shown here in cyan and magenta.

Another advantage of the designed co-crystal is when we convert the DNA-DNA junction with a weak blunt end junction to a programmed junction driven by sticky base overhangs, shown here with a 2-base overhang.

Using our LEGO representation, we are going to switch colors to show you our engineered version of the scaffold protein with black and yellow. Just like the original interactions, the proteins bind the DNA in a lattice orientation. A key difference in the DNA blocks is the new stronger connections depicted by the grey linkers.

They build out to become a sturdier, robust crystal with nice large pores for application.

One of the niftiest aspects of these expanded crystals is the modularity – we can put any sequence we want for the DNA struts. With the inserts shown in dark green, we could grow crystals with strut sequence A to capture a transcription factor of interest for structure determination, or sequence B to capture an enzyme of interest for catalysis, or sequence C to temporarily capture a fluorescent protein for biosensing.

We will explore all of these applications in the future at Colorado State University.