Biomod/2011/Harvard/HarvarDNAnos:Results Box

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Rectangular Box Container Results

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Overview

  • We have designed and folded two robust origami structures: the lid and the barrel. These two structures are the components of our box.
  • We demonstrate that we can close and open the box with high efficiency.
  • We demonstrate that we can attach AuNP cargo to our lids and/or our barrels and then close the box, placing the AuNP cargo within the box.
  • We demonstrate that we can photocleave the AuNP cargo off of our lids, solubilizing the cargo.
  • Although we have not directly shown that our closed boxes can contain soluble cargo and thus release soluble cargo upon opening, we have verified all of the system's components: we can close the box, we can load the box with cargo, we can solubilize the cargo, and we can open the box.
    • To fully reach our goal, we now only need to photocleave our Lid-AuNP complexes after forming closed boxes via introduction of barrel. If TEM of this photocleaved sample yields closed boxes with contained gold, this demonstrates that our box can securely hold our cargo. Subsequent introduction of the key signal should open the box and release our cargo into solution.

Folding Origami

Gel

This 1.5% agarose gel indicates that the barrel and lid structures formed. The lid runs slightly faster than the barrel, as expected due to its smaller size. Both structures run faster than M13mp18 scaffold, likely because they are more compact.

The Barrel

This TEM image confirms that the barrels folded correctly. The dimensions are roughly as we designed them. Note that some barrels are standing upright while others are laying on their sides. The upright ones appear as white squares with a hollow, dark square center. The ones that are laying on their sides appear as white rectangles with a dark rectangular middle that stretches lengthwise across the entire middle of the rectangle; this is because the electron beams must pass through fewer layers of DNA to go through the middle of the sides and more layers of DNA to go through the edges of the sides.

The Lid

This TEM images confirm that the lids folded properly as well. The dimensions are roughly exactly as we designed them. Note that, for an unknown reason, lids deposited on TEM grids favor standing upright on their edges rather than laying flat on their tops and bottoms.
On AFM mica discs, however, the lids do prefer laying flat.

Opening and Closing the Box

Closing the (Empty) Box

Combining a solution of lid with a solution of barrel results in the formation of closed boxes. The three closed boxes shown in this TEM image are laying on their sides, with the lids standing upright. Note that our definition of "closed box" is a barrel that has associated with at least two lids that are both in the correct orientation, i.e. are covering the barrel's openings.
This TEM image of a lone closed box shows that the dimensions of the boxes approximately match what our design specified.
This TEM image showcases the variety of structures that form when lids and barrels are mixed. While the majority of structures are closed boxes, a small proportion are barrels associated with only one lid or with lids in the wrong orientation. Because we mixed barrels with an excess of lids, our closed box solutions do contain many extra, non-used lids.
The vast majority of closed boxes when deposited on a TEM grids take on an orientation where the barrel is laying on its side and the lids are thus standing upright. However, it is also important to be able to spot the alternate orientation, with the barrel upright and the lids parallel to the grid. In this image, note that there is a raised, square region "underneath" and slightly larger in area than the barrel. The dimensions of this square match the dimensions of a lid laying flat on its back. Unfortunately, it is difficult to conclude whether this barrel is associated with one or two lids due to the indirectness of transmission electron microscopy.
AFM imaging of a solution of closed boxes yields an abundance of cubical species.

Opening the Box

Before addition of key strands, there is an abundance of closed boxes.

After addition of the key strands, however, nearly all barrels and lids detach from one another, demonstrating that we can controllably close and open this box.

Gel

Running an agarose gel allowed us to extract bands corresponding to lid:barrel and lid:barrel:lid complexes. The gel also shows that boxes are opened effectively by addition of key strand.
TEM image of Lid:Barrel gel extract.
TEM image of Lid:Barrel:Lid gel extract.

Loading the Box

Lid-AuNP Complexes

Introducing to the lid an excess of 5 nm gold nanoparticles with conjugated ssDNA complementary to the lid handle strand yields Lid-AuNP complexes.
The positioning of the cargo on the lid is as we expected.
The lid continues to orient itself in two ways: flat and standing up.

Lid-AuNP/Barrel Complexes

Further addition of barrels to the above Lid-AuNP complexes gives a small amount of closed boxes with gold apparently positioned inside the walls of the box.

Barrel-AuNP Complexes

Our yield of Lid-AuNP/Barrel complexes was rather low, probably because of our yield of Lid-AuNP complexes (above) was rather low. Therefore, we decided to also attach gold cargo to the inside of our barrels.

We found that the efficiency of loading cargo onto the barrels was higher than loading cargo onto the lids, despite the fact that the gold must find its way into the barrel in order to attach. This may be due to post-binding entropic effects or because there are three handles inside our barrels whereas only one handle on our lids. Note that some of our barrels actually contain more than one AuNP.

Lid-AuNP/Barrel-AuNP Complexes

Mixing Lid-AuNP complexes with Barrel-AuNP complexes gives a much higher yield of closed boxes with contained gold! Success! Notice that some of our cargo is bound to the barrel and some of our cargo is bound to the lid.

Releasing Cargo

Photocleavage of Lid-AuNP Complexes

Previously, we showed that our photocleavage protocol can successfully cleave ssDNA ordered from IDT with an internal photocleavable spacer. We use these PC spacers as a linker between the lid and the lid's gold handle when we want to controllably release our nanocargo. Above, we showed that we can attach gold to lids that are not equipped with a PC spacer. Here, we show that we can also attach gold to lids that are equipped with a PC spacer.

Prior to photocleavage, note the presence of Lid-AuNP complexes, indicated by the black boxes.

However, after photocleavage, there exist essentially no Lid-AuNP complexes: we have thus demonstrated that we can controllably attach and release gold from our lids.

Solubilization Within the Box and Subsequent Opening of Box to Release Cargo

Future steps:

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