The Chaput laboratory studies fundamental and applied problems at the interface of chemical and synthetic biology.
Synthetic biology encompasses a wide range of molecular biology tools that enable researchers to precisely manipulate the DNA sequence within a gene, gene cluster, or genome. Recent developments, however, have made it increasingly possible to generate man-made enzymes that can perform similar functions on artificial genetic polymers that are distinct from those found in nature (DNA and RNA). Collectively referred to as xeno-nucleic acids, or XNAs, these genetic polymers have unique physicochemical properties that include resistance to nuclease digestion and expanded chemical functionality. We envision a future where many of the same synthetic biology tools available to manipulate DNA and RNA are available to manipulate XNA. Such efforts open the door to a vast new world of synthetic genetics, where artificial genetic polymers can be used to create new tools for biotechnology and medicine, and possibly even improve our understanding of the origin of life itself.
While the possibility of manipulating XNAs in a test tube has enormous potential for new applications in biotechnology and medicine, several challenges must be overcome before such achievements can be realized. The most significant challenges include:
- Establishing new chemical synthesis strategies that produce XNA monomers on the gram to multi-gram scale and expand the chemical functionality of XNA beyond the natural bases of adenine (A), cytosine (C), thymine (T), and guanine (G).
- Designing new molecular evolution approaches that facilitate the production of XNA enzymes that can recognize and modify XNA substrates with high catalytic efficiency.
- Developing automated approaches that enable the rapid discovery of XNA aptamers and XNA catalysts to a broad range of biologically important targets.
- Elucidating the molecular structures of XNA enzymes and in vitro selected XNA aptamers and XNA catalysts to high resolution.
Chemical Synthesis
In order to develop enzymes that function on artificial genetic polymers, researchers must have access to XNA substrates (nucleotide triphosphates and oligonucleotides) that can be produced on the scales required for enzyme evolution and eventual downstream applications. This challenging endeavor requires establishing new chemical synthesis strategies that enable the production of XNA monomers on the multi-gram scale. In addition, new methodologies are needed to synthesize XNA substrates with expanded chemical functionality.
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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
Synthetic genetics will require a wide range of enzymes that can modify XNA substrates in different ways. Of these, polymerases represent an important initial function due to their ability to synthesize and replicate genetic information. To help meet this challenge, we have developed a general strategy for evolving XNA polymerases in vitro. Our technology, referred to as droplet-based optical polymerase sorting (DrOPS), employs an optical sensor to monitor polymerase activity inside the microenvironment of uniform synthetic compartments generated by microfluidics.
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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)
In vitro selection is a powerful method for evolving nucleic acid molecules with specific target binding affinity or catalytic activity. Unfortunately, natural genetic polymers have limited utility in applications that require high biological stability, as these polymers are rapidly degraded by endogenous nucleases. By comparison, most XNAs are resistant to nuclease digestion, making them valuable reagents for diagnostic and therapeutic applications that require high biological stability.
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- Darwinian Evolution of an Alternative Genetic System Provides Support for TNA as an RNA Progenitor
- Reverse Transcription of Threose Nucleic Acid by a Naturally Occurring DNA Polymerase
- Evaluating TNA Stability Under Simulated Physiological Conditions
Structure Determination
The evolution of polymerases that can synthesize and recover genetic information stored in XNA polymers demonstrates that the biology concepts of heredity and evolution are not limited to the natural genetic polymers of DNA and RNA. 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.
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