Description:
OBJECTIVE: Design and develop DNA origami-based bio-scavengers with high affinity for organophosphorus compounds and demonstrate these systems can be optimized for use in the molecular sequestration of nerve agents. DESCRIPTION: The DOD has the need for a universal organophosphorus (OP) scavenger that will protect against multiple OP compounds, including all existing nerve agents. The ideal scavenger should be rapid, irreversible, and specific and have a prolonged circulation time in the bloodstream. The ideal scavenger should also be biologically innocuous in the absence of OP compounds. In particular the optimum scavenger should not stimulate an immune response. Scavengers can also be used prophylactically by inactivating OP compounds before they can react with the target acetcylcholinesterase (AChE). In biological systems, avidity is often used to describe the combined strength of multiple bond interactions. Avidity is distinct from affinity, which is a term used to describe the strength of a single bond. Avidity is the combined synergistic strength of bond affinities rather than the sum of bonds. Avidity is commonly applied to antibody interactions in which multiple binding sites simultaneously interact with a target molecule. When many binding interactions are present at the same time, transient unbinding of a single site does not allow the molecule to diffuse away, and binding of that site is likely to be reinstated. It has been demonstrated multiple binding sites can increase the effective binding of a molecular species by up to five orders of magnitude relative to the affinity of any univalent bond within the system. A DNA origami structure may have up to 250 binding sites on a single moiety. This presents a powerful platform for molecular sequestration. Current treatment of OP poisoning involves a combination of therapies. Drugs such as atropine are administered to counteract the effects of acetylcholine. An oxime such as pralidoxime reacts with the inhibited cholinesterase, removing the organophosphate from cholinesterase. The cholinesterase is then able to resume normal function. Anticonvulsant drugs such as diazepam are used to control tremors and convulsions. These therapies are effective in preventing death. However, performance deficits, behavioral incapacitation, loss of consciousness, and potential permanent brain damage can occur in the absence of further treatment. An effective bioscavenger that can reduce the concentration of all OP"s to low levels (less than one micromolar) in a short period of time (less than 2 minutes) would provide DOD personnel with needed protection from the harmful defects of nerve agent exposure. Intravenous (IV) plasma derived butyrylcholinesterase (hBuChE) is a very effective bioscavenger that has been shown to provide protection from lethal exposure to nerve agent using animal models. However, hBuChE is logistically difficult to use in battle field scenarios since it typically requires refrigeration. Attempts have been made to lyophilize hBuChE. However, reconstituted hBuChE shows a considerable loss in activity. Furthermore, hBuChE is perishable, and a constant supply can only be maintained with on-going blood donations. Butryrlcholinesterase can also be produced using recombinant methods (rBuChE). However, rBuChE has been shown to be less effective than hBuChE. In general protein folding is unpredictable and difficult to control. Lyophilization and reconstitution can produce unpredictable results. Proteins are perishable and tend to be very immunogenic. Hence, a non-protein-based bio-scavenger may provide significant advantages for treating nerve agent exposure. Recent advances in DNA-based origami and hybrid DNA-based nanostructures that combine origami construction with designer motifs offer a new bio-technological pathway to man-engineered bio-scavengers. DNA origami is the folding of DNA strands into 2D or 3D, and DNA motifs may be utilized to array individual DNA origami units into even larger and more complex bio-nanostructures. In a DNA origami, a long single-stranded plasmid DNA, like M13mp18 ssDNA, can be folded into a space-filling curve and held in place with shorter"staple"strands, which crosslink and stabilize the structure, enabling the formation of complex 2D and 3D shapes. The molecular weight of a typical DNA origami structure (e.g., based on M13mp18) can be over 2M daltons with a relatively large surface area, which can be decorated with multiple precisely addressed molecular recognition units. Theoretically, each staple strand can attach to one molecular recognition unit, which will allow for the capture of up to 250 target molecules by each individual DNA origami construct, and this number can be multiplied using DNA motif-based arraying techniques. Therefore, DNA origami-based platforms can present large numbers of molecular sequestration sites. Furthermore, such DNA-based bio-scavengers would not elicit a strong immunogenic response. This is in contrast to proteins where strong immunogenic responses are normal. A strong immunogenic response to a scavenger can have very negative implications. For example, a person can become allergic to cholinesterase-like molecules that can lead to autoimmune-like symptoms. In general, the design of large and diverse DNA-based nanostructures using naturally occurring genetic components (i.e., using only four base pairs) involves a great deal of computational complexity. Indeed, the task of determining the DNA sequence sets required for the self-assembly of highly optimized and stable geometrical forms presently requires the use of specialized algorithms and distributed computing. While active DOD programs are presently advancing the state of the art in design automation software for DNA architectures, the design and associated synthesis problem can be greatly simplified through the use of expanded DNA alphabets such as AEGIS (Artificially Expanded Genetic Information System) that uses twelve nucleic acids in its genetic code. These artificially expanded DNA systems offer many strategic advantages (e.g., managing non-canonical forms, more chemically stable, etc.) and can be used to realize fundamental building blocks that allow for the additional of clickable functionality (i.e., which could be useful for defining electro-optical diagnostic techniques for monitoring molecular sequestration action). The chances of an adverse immune reaction would also be further reduced through the use of AEGIS type components since the artificial bases do not exist in nature; it is unlikely that a person will have future encounters with the artificial structure produces by these bases. Hence, DNA origami-based platforms that utilize hybrid building blocks represent a promising pathway to a novel class of bio-scavengers for nerve agent sequestration. PHASE I: Formulate a complete design for a bio-scavenger based upon DNA origami components that sequesters organophosphorous compounds from blood using integrated molecular capture. It is also strongly recommended that the DNA origami-based platform be specified so as to incorporate electro-optical based diagnostic monitoring of the sequestration process. The scavenger should reduce OP"s concentrations to low levels (less than one micromolar) in a short period of time (less than 2 minutes). The scavenger should remain in the bloodstream for an extended period of time (at least 10 days) after administration at levels that provide protection from OP"s. The scavenger should be non-immunogenic. The scavenger should be lyophilizable and remain active after reconstitution. The lyophilized form of the scavenger should be stable for extended periods of time (at least 2 years at temperatures not exceeding 50 degrees C). Demonstrate proof of concept and feasibility using candidate OP binding moiety testing. For demonstration purposed, is can be assumed that the initial OP concentration can be as high as one millimolar. An in vitro study of a pesticide or other nerve agent simulant can be used for proof-of-principle in Phase I. PHASE II: Develop and test a DNA origami-based bio-scavenger that achieves the functionality and performance requirements specified in the Phase I requirements. It is expected that the bio-scavenger will first be tested using OP pesticides and/or nerve agent simulants. The bio-scavenger should also be tested against bona fide OP nerve agents. Since testing with OP nerve agents is required during Phase II development, and as small business firms are unable to handle and utilize chemical weapon agents (CWAs), a testing facility approved to utilize these materials must be identified (it strongly recommended that a qualified DOD laboratory or appropriately licensed commercial laboratory be identified for use in testing against the OP nerve agents). PHASE III: Research and development during Phase III efforts will be directed toward refining final deployable designs for OP scavengers. In vivo safety and efficacy testing in animal models will be performed. Design modifications based on results from tests conducted during Phase II will be incorporated. Manufacturability specific to the Joint Chemical and Biological Defense Program CONOPS and end-user requirements should be examined. Molecular scavengers will have numerous commercial applications, particularly in the field of medicine.
