The Chaput lab is working to develop the reagents and tools needed to synthesize and manipulate artificial genetic polymers (XNAs) in a manner analogous to the way that DNA and RNA are routinely synthesized and modified in labs across the globe.
The biotechnology revolution is built on the simple concept that enzymes found in nature can be used to improve human health. Using these gifts, scientists have assembled a molecular biology toolkit consisting of polymerases, ligases, kinases, and nucleases. The sophistication of these tools and the advances made with them now begs the question: What new technologies could we develop or what new discoveries could we make, if we were no longer constrained to the natural genetic polymers of life?
To address this question, we take a convergent science approach that combines the scientific disciplines of chemistry, engineering, and molecular and structural biology.
Specific projects include:
Chemical Synthesis
Chemistry students in the lab are working to develop new types of XNA monomers and investigating strategies for improving the functional properties of XNA through added chemical functionality. In addition, they are also seeking ways to establish XNA polymers with an expanded genetic alphabet that could be used in future synthetic biology applications.
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- A Scalable Synthesis of α-L-Threose Nucleic Acid Monomers
- A Gram-Scale HPLC-Free Synthesis of TNA Triphosphates Using an Iterative Phosphorylation Strategy
Enzyme Engineering
Enzyme engineering students in the lab have established a powerful microfluidic platform for evolving DNA modifying enzymes. The goal of these studies is to rapidly evolve naturally occurring enzymes with the ability to recognize XNA substrates. These efforts are producing the tools needed to evolve XNA affinity reagents (aptamers) and catalysts for practical applications in molecular medicine.
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- A General Strategy for Expanding Polymerase Function by Droplet Microfluidics
- Improving Polymerase Activity with Unnatural Substrates by Sampling Mutations in Homologous Protein Architectures
In Vitro Selection (SELEX)
Molecular biology students interested in evolving functional XNAs are working to discover XNA molecules with specific target binding affinity or catalytic activity. XNA molecules produced from these selections are valuable reagents for diagnostic and therapeutic applications as they are completely resistant to degradation by biological nucleases that rapidly degrade DNA and RNA.
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- Darwinian Evolution of an Alternative Genetic System Provides Support for TNA as an RNA Progenitor
- Synthesis and Evolution of a TNA Aptamer Bearing 7-Deaza-7-Substituted Guanosine Residues
- Evaluating TNA Stability Under Simulated Physiological Conditions
Structure Determination
Structural biology students in the laboratory are working to solve the X-ray crystal structures XNA enzymes developed by the protein engineering team. Examining how these enzymes function at a molecular level is necessary for understanding their mechanism of action and guiding the design of new XNA enzymes that function with enhanced activity. In addition, these students are also pursuing the structures of in vitro evolved XNA aptamers and catalysts, which will provide new insights into biopolymer folding.
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