Students in the Chaput lab are working to develop the enzymes, reagents, and tools needed to synthesize, propagate, and evolve artificial genetic polymers (XNAs) with new physicochemical properties.
This is a highly interdisciplinary research program that includes organic chemistry, enzyme engineering, droplet microfluidics, and X-ray crystallography.
The long-term goals of our research are to:
- study fundamental questions related to how biopolymers fold and function
- investigate the mechanism of DNA polymerases
- establish new routes to diagnostic and therapeutic agents
- expand the toolkit for synthetic biology
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
- P(V) Reagents for the Scalable Synthesis of Natural and Modified Nucleoside Triphosphates
Enzyme Engineering
We 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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- Elucidating the determinants of polymerase specificity by microfluidic-based deep mutational scanning
- Fluorescence-activated droplet sorting for single-cell directed evolution
- A General Strategy for Expanding Polymerase Function by Droplet Microfluidics
Therapeutic & Diagnostic Agents
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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