April 06, 2012

DNA origami hinged barrel with logic gates for programmable delivery of drugs

Researchers at Harvard University have developed a robotic device made from DNA that potentially could seek out specific cell targets within a complex mixture of cell types and deliver important molecular instructions, such as telling cancer cells to self-destruct.

A research team headed by George Church used what they call the DNA origami method, in which complex three-dimensional shapes and objects are constructed by folding strands of DNA. In this case, the researchers created a nanosized robot in the form of an open barrel whose two halves are connected by a hinge. The DNA barrel, which acts as a container, is held shut by special DNA latches that can recognize and seek out combinations of cell-surface proteins, including disease markers. When the latches find their targets they reconfigure, causing the two halves of the barrel to swing open and expose its payload. The container can hold various types of payloads, including specific molecules with encoded instructions that can interact with specific cell surface signaling receptors.

Science - A Logic-Gated Nanorobot for Targeted Transport of Molecular Payloads

We describe an autonomous DNA nanorobot capable of transporting molecular payloads to cells, sensing cell surface inputs for conditional, triggered .activation, and reconfiguring its structure for payload delivery. The device can be loaded with a variety of materials in a highly organized fashion and is controlled by an aptamer-encoded logic gate, enabling it to respond to a wide array of cues. We implemented several different logical AND gates and demonstrate their efficacy in selective regulation of nanorobot function. As a proof of principle, nanorobots loaded with combinations of antibody fragments were used in two different types of cell-signaling stimulation in tissue culture. Our prototype could inspire new designs with different selectivities and biologically active payloads for cell-targeting tasks.

43 pages of supplemental material

AFM micrograph of closed nanorobots. Nanorobots were diluted to ~10 nM into TAE buffer containing 12 mM Mg2+. 5 μL were applied directly on freshly-cleaved grade V mica mounted on a sample plate using optical adhesive no. 61. Samples were visualized in a Veeco/Bruker Multimode Nanoscope V in fluid tapping mode, using SNL-10 C probes.

Open barrel design

We chose an open barrel design for the nanorobot because we expected it allow for the production of the highest yield of functional nanorobots for subsequent experimentation. An open design allows for single-step loading of cargo: once the origami is folded and purified from excess staple strands, the DNA-anchor-linked cargo is simply added in excess and allowed to diffuse into the nanorobots and bind. We thus avoid extra preparation steps that may be required of a closed design, such as closing the container after loading it in an open state, or freeing a tethered cargo after loading/activation. This strategy does limit our to payloads that can be linked to DNA, but this is not a problem in our desired application of stimulating cell signaling, because antibody payloads remain active even while attached to the nanorobot.

We estimate that approximately 2000 particles per second diffuse through the open ends of the nanorobot, so the efficiency of cargo loading is unlikely to be diffusion limited on the time scales we used for loading (~24 hrs).

3D Models highlighting guide staples. (a) guide staples (red) bridge top and bottom domains of the nanorobot. 8-base toeholds are included to allow removal by addition of fully-complementary strands after folding and purification. (b) After guide staple removal, only aptamer locks hold the structure in the closed state.

The investigators used this system to deliver instructions, which were encoded in antibody fragments, to two different types of cancer cells – leukemia and lymphoma. In each case, the message to the cell was to activate its "suicide switch" – a standard feature that allows aging or abnormal cells to be eliminated. And since leukemia and lymphoma cells speak different languages, the messages were written in different antibody combinations.

This programmable nanotherapeutic approach was modeled on the body's own immune system in which white blood cells patrol the bloodstream for any signs of trouble. These infection fighters are able to home in on specific cells in distress, bind to them, and transmit comprehensible signals to those cells to self-destruct. The DNA nanorobot emulates this level of specificity through the use of modular components in which different hinges and molecular messages can be switched in and out of the underlying delivery system, much as different engines and tires can be placed on the same chassis. The programmable power of this type of modularity means the system has the potential to one day be used to treat a variety of diseases.

"We can finally integrate sensing and logical computing functions via complex, yet predictable, nanostructures – some of the first hybrids of structural DNA, antibodies, aptamers, and metal atomic clusters – aimed at useful, very specific targeting of human cancers and T-cells," said Dr. Church.

Because DNA is a natural biocompatible and biodegradable material, DNA nanotechnology is widely recognized for its potential as a delivery mechanism for drugs and molecular signals. There have, however, been significant challenges to its implementation, such as what type of structure to create; how to open, close, and reopen that structure to insert, transport, and deliver a payload; and how to program this type of nanoscale robot.

By combining several novel elements for the first time, the new system represents a significant advance in overcoming these implementation obstacles. For instance, because the barrel-shaped structure has no top or bottom lids, the payloads can be loaded from the side in a single step without having to open the structure first and then reclose it. Also, while other systems use release mechanisms that respond to DNA or RNA, the novel mechanism used here responds to proteins, which are more commonly found on cell surfaces and are largely responsible for transmembrane signaling in cells. Finally, this is the first DNA-origami-based system that uses antibody fragments to convey molecular messages – a feature that offers a controlled and programmable way to replicate an immune response or develop new types of targeted therapies.

Research that references the DNA nanobot work

A mechanical Turing machine: blueprint for a biomolecular computer

We describe a working mechanical device that embodies the theoretical computing machine of Alan Turing, and as such is a universal programmable computer. The device operates on three-dimensional building blocks by applying mechanical analogues of polymer elongation, cleavage and ligation, movement along a polymer, and control by molecular recognition unleashing allosteric conformational changes. Logically, the device is not more complicated than biomolecular machines of the living cell, and all its operations are part of the standard repertoire of these machines; hence, a biomolecular embodiment of the device is not infeasible. If implemented, such a biomolecular device may operate in vivo, interacting with its biochemical environment in a program-controlled manner. In particular, it may ‘compute’ synthetic biopolymers and release them into its environment in response to input from the environment, a capability that may have broad pharmaceutical and biological applications.

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