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A new four-year, multi-million dollar award from the Defense Advanced Research Projects Agency (DARPA) will be used to develop the technology necessary to synthesize, screen and sequence artificial genetic polymers composed of threose nucleic acid (TNA).
John Chaput, a professor in the Department of Chemistry and Biochemistry and research investigator in the Biodesign Institute will lead ASU’s effort to evolve TNA molecules that fold into novel 3-D shapes with ligand binding affinity and catalytic activity.
Chaput is joined by ASU colleague Wade Van Horn, Department of Chemistry and Biochemistry, as well as Martin Egli, Department of Biochemistry at Vanderbilt University, and Jennifer Heemstra, Department of Chemistry, University of Utah.
The research is part of a new DARPA program called Folded Non-Natural Polymers with Biological Function (Fold F(x)), which plans to use synthetic polymers to address rapidly emerging health and defense threats.
Naturally occurring biopolymers like DNA, RNA and proteins are limited to functions that are required to sustain life, and prone to degradation by metabolic pathways that recycle biomolecules for other purposes. Recognizing these limitations, researchers would like to design new types of synthetic polymers, with versatile functions and folding motifs, that are stable to biological and harsh environmental conditions.
“This project integrates chemistry with molecular biology and genetics to produce synthetic molecules with tailor-made activities,” Chaput says.
The group's project is based on Chaput’s efforts to develop TNA as an artificial genetic polymer that is capable of heredity and evolution. This requires using organic chemistry to synthesize TNA monomers that are not commercially available, and engineering DNA polymerases to copy genetic information back and forth between TNA and DNA.
By introducing a selective amplification step into the replication cycle, like the ability to bind a small molecule target or catalyze a chemical reaction, large combinatorial pools can be searched for TNA molecules with desired functional properties.
This process is analogous to natural selection, where deleterious traits are removed from the population through iterative rounds of selection and amplification. In the case of TNA, molecules that meet the selective challenge will be recovered and amplified to generate progeny molecules that increase in abundance.
Affinity reagents, like those under development, may prove invaluable for biomedical applications. Their strength lies in their foreignness. Most diagnostic tests currently use antibodies as the affinity reagent. However, antibodies are not stable to heating, and can be degraded by enzymes present in biological samples.
In contrast, TNA affinity reagents are expected to remain functional after heating and cooling, and biological systems have not developed the kinds of enzymes necessary to recognize and degrade these synthetic polymers. A key aspect of the project is new technology that will quickly generate affinity reagents capable of converting the presence of a specific molecule into an optical signal. Heemstra says, “Directly selecting for TNA affinity reagents that have the desired optical output will enable us to rapidly respond to emerging threats.”
The team plans to determine the 3-D structure of functional TNA molecules isolated by in vitro selection. Egli will head the X-ray crystallography experiments and Van Horn the nuclear magnetic resonance spectroscopy efforts. Understanding the structural architecture of TNA is an important part of the overall project because very little is known about the atomic organization of TNA. Structural information gained from these studies will allow scientists to understand how unnatural polymers, like TNA, fold into globular structures.
“Since TNA has a different backbone than DNA and RNA, it will be interesting to learn what shapes these molecules can adopt. This structural information will help us to understand how TNA molecules perform more complex functions as biosensors and catalysts,” Van Horn says.
The new project will also introduce modified bases into TNA molecules. The combination of altered bases and threose backbone may yield novel folding geometries, with as-yet unknown functional properties, potentially allowing nucleotides to perform in a manner similar to proteins.
The technical challenges involved in the current project are formidable, and therefore consistent with the high-risk, high-reward model of DARPA-funded research. The team hopes the synthetic biology initiative will be a springboard for new ideas and techniques to be exploited by other labs and, in time, brought to the forefront of molecular biology.
The Department of Chemistry and Biochemistry is an academic unit in ASU's College of Liberal Arts and Sciences.