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.
- A Scalable Synthesis of α-L-Threose Nucleic Acid Monomers
- A Gram-Scale HPLC-Free Synthesis of TNA Triphosphates Using an Iterative Phosphorylation Strategy
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.
- A General Strategy for Expanding Polymerase Function by Droplet Microfluidics
- Improving Polymerase Activity with Unnatural Substrates by Sampling Mutations in Homologous Protein Architectures
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.
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.