Building really small things is typically extremely hard; however since the development of the DNA origami technique a new world of opportunity has been opened. DNA origami allows scientists to relatively easily build arbitrary shapes and 3-dimensional structures on the nanoscale – some examples of these are shown below.
For more information of the DNA origami technique see DNA design. There are many applications for this technology; however one of the most interesting opportunities which emerged early on was the prospect of developing nanoscale vessels which could deliver molecular cargo. The series of events unfolds as follows: a vessel is loaded with the cargo, the vessel is delivered, and then a release mechanism unloads the cargo.
DNA origami has been used to not only just capture and release cargo, but to autonomously target selective cells and release the contents directly to the target site via logic-gated mechanisms. Some of these designs include Douglas et al. (top) and Ranjbar et al. (bottom).
DNA origami is an excellent means for developing targeted delivery vessels as the surfaces are fully addressable by conjugation to the staple strands. This allows origami structures to be modified with ligands, antibodies, hormones, labels for bioimaging and so on with atomic precision.
By housing a drug inside a DNA origami vessel and modifying the outer surface with receptors one can facilitate selective interaction with cells. Coupled with a logic-gated release mechanism DNA origami structures can directly unload a payload to a target site.
Chemotherapy for conditions such as cancer and tuberculosis relies on the use of non-specific poisons to kill harmful cells. It works, but not without significant collateral damage to healthy cells.
Targeted delivery vessels are a valuable technology which can not only protect the body from the toxic effects of the pharmaceuticals en route to the site, but can also deliver an improved response per dose. The vessels can also be used for many other means, such as protecting a precious of fragile payload from a potentially hostile medium.
Logic-gated vessels with various medicinal cargo and selective release mechanisms could potentially in the future be developed for some sort of system resembling an artificial immune system, capable of detecting various cues from the environment and releasing the appropriate payload.
The Problem (Relevance)
The process of targeted delivery can be broken down into three steps: loading the vessel with the drug of interest, recognising the target site, and releasing the payload directly at that site. As shown above with Douglas et al. and Ranjbar et al., effective vessels have already been developed, but thus far the method of loading the cargo is limited to chemically binding it to the vessel.
What if we could load a vessel with an unbound payload? So that when delivered rather than remaining attached to the vessel the cargo is free to diffuse out and perform its function. Would the free cargo be more effective compared to a bound cargo? Could it fit into smaller spaces and perform unique tasks that were previously unachievable due to being bound to the vessel?
What if we could take it a step further and build a vessel which was selectively responsive to autonomously loading a particular cargo? Could that lay the foundation for a new generation of smart drug delivery vessels which can not only selectively unload their cargo, but can also identify free cargo in solution and load it?
Our Solution (Merit)
These are interesting questions to ask, but for the sake of practicality they should be considered one at a time. First, we needed to ask if it were possible to capture any molecular cargo unbound inside a vessel.
Capturing unbound cargo isn’t a trivial task. Resorting to probability and locking a vessel at an arbitrary time and hoping to capture a particle is a poor strategy due to the enormous dispersity of particles in solution relative to the confined cavity space of most vessels.
The only alternative, we found, was to design a vessel which could actually be triggered to close by the proximity of the target molecule. After hundreds of bad drawings and a few late nights on caDNAno, out popped our design (which ended up looking a lot like a mousetrap.)
Our vessel consists of two hollow DNA origami lids joined by a spring-loaded hinge. The lids are braced open against the torsional strain of the hinge, and the brace contains a cut-site for a specific endonuclease. The endonuclease lands on the box, it cuts the brace, and then the lids slam shut and capture it. The endonuclease can also be engineered to carry other molecules such as proteins with it while it cuts.
This is a good solution because it proposes a way to potentially capture a specific unbound molecular cargo inside of a DNA origami vessel, which due to the nature of DNA origami structures could be easily modified with receptors to mediate targeting to diseased cells and deliver a payload.
Even with the application of targeted delivery aside, capture of free molecules inside of molecular cages has proved to be a significant challenge and this project offers a novel solution. This design also creates opportunity for research and discovery regarding the mechanical properties of spring-loaded DNA origrami structures, such as torsional and longitudinal stiffness.
Project Goals (Specification and Feasibility)
There is a trade-off between choosing goals that are realistic in the short timeframe of BIOMOD and which are also scientifically significant and exciting. The prospect of developing new DNA origami vessels which can selectively capture unbound molecular cargo is one which can be explored in many directions, but for this competition we have chosen strategic goals which allow us to test the core concept and its feasibility.
Criteria For Success (Checklist)
Clicking the links below will lead you to the results of each experiment.
- Design box with functional components
- Structural characterisation
- Functional characterisation (hinge, braces, latches)
- Design a payload which can trigger the closing mechanism and be caught inside the vessel
The Ideal Goal For This Project
The end goal of the project is to design, synthesise and verify a DNA origami vessel which can capture an endonuclease containing payload unbound inside the cavity (within size constraints.) In succeeding we will have pioneered a novel method for reliably DNA origami vessels with unbound cargo.
Our Goals For BIOMOD
BIOMOD is too short to realistically achieve everything we have stated above, and so we have three primary goals which we wish to achieve as a proof of concept for this mechanism:
- Demonstrate that a DNA origami structure can be braced open and triggered to close
- Demonstrate that our engineered payload is functional and can cut the brace
- Demonstrate that our payload can be caught inside the vessel by first covalently binding
To achieve those two points we have made some alterations to our design that would not be present in the ideal end goal:
- We have designed our vessel with two braces instead of one for extra stability (it was uncertain if a single brace could withstand the torque of the hinge due to limited literature on mechanical characteristics of spring-loaded DNA origami)
- The payload first covalently binds to the inside of the cavity so that it can sequentially cut the braces and be caught inside (this is necessary due to the design containing two braces)
If we can demonstrate sequential cutting of braces and capture of the bound payload, we could then move on to a new design with a single, strategically placed brace.
The purpose of our project in the BIOMOD competition isn’t to capture a payload of any medicinal value, but rather to prove the concept that a payload can be captured unbound inside of a DNA origami box which could potentially be modified to facilitate targeted delivery.
There a number of potential directions to take this project from here on. Some interesting avenues and opportunities include:
Testing different brace-releasing mechanisms other than via endonuclease
Cutting the DNA brace with a restriction enzyme is only one way of releasing the pressure and capturing a payload. For example perhaps a binding event could take place in which the DNA in the brace binds to an accessory on the payload and thus stops supporting the lids causing the box to collapse.
This is worthwhile exploring as it may offer a more elegant solution with less or perhaps no modification to the payload of interest in order to capture it. Different variations of brace structures and receptors could exploit natural properties of the payload of interest to trigger selective closing.
Potential cargo release mechanisms
The next step is obviously to modify or design a new spring-loaded structure which can not only trap but also release the molecular payload. A couple of potential strategies include:
- Star activity of the endonuclease could eat out of the origami at body temperature. Perhaps the origami could be ingested in a frozen tablet to strategically time the degradation.
- Addition of surface receptors for internalisation of origami into T-cells or otherwise where the harsh environment will degrade the origami and release the payload.
- Reformation of the brace at the target site.
- Destruction of the hinge region to separate the lids.