Development of a Fully Enzymatic Oligonucleotide Synthesis Cycle by Engineered Template Independent Polymerases and a Novel Phosphate dNTP Blocking Group

Project Summary/Abstract DNA synthesis has revolutionized the field of synthetic biology, leading to new therapeutics, bio-based fuels and chemicals, and materials. The chemical method to synthesize DNA was developed over 30 years ago and is still challenged by high costs and limits in DNA length (andlt;200 nucleotides). As synthetic biology has outpaced current DNA synthesis technology, the scope of many research approaches is now limited by cost and length of synthesized DNA. Enzymatic DNA synthesis approaches employ polymerase enzymes and stepwise incorporation and deprotection of blocked nucleotides (dNTPs) and are a promising alternative to overcome the limitations of chemical DNA synthesis. Despite their potential, most enzymatic approaches still rely on chemical treatment steps to remove blocking groups from the synthesized sequence. Chemical deblocking steps can produce hazardous waste and repeatedly subject oligonucleotides to degradative chemicals. In this Phase I SBIR proposal, Molecular Assemblies Inc. proposes to develop a fully enzymatic DNA synthesis approach. This approach has at its core three key enzymatic steps: 1) polymerase incorporation of 3′-O-blocked nucleotides, 2) an enzymatic deblocking step to remove the phosphate blocking group from the 3′-hydroxyl, and 3) a novel enzymatic clean-up to deplete unreacted material. By utilizing the efficiency and specificity of enzymatic rather than chemical processes, we seek to develop an environmentally friendly DNA synthesis approach with the goal of generating longer (andgt;200 nucleotides), purer DNA. One key target of the proposed work is to engineer the template-independent polymerase, Terminal deoxynucleotidyl Transferase (TdT), for improved 3′-O- phosphate dNTP incorporation. We will couple 1) rational design of amino acid mutations using the protein design software, Rosetta, and 2) in silico bioprospecting to produce screening libraries comprising phylogenetically diverse TdT backgrounds. This combined enzyme engineering approach has great potential to identify enzyme mutants with distinct phenotypes. We will express and screen the resulting targeted libraries using our established high-throughput nucleotide incorporation assays to identify the most active TdT variants. We will then optimize the enzymatic clean-up and deblocking steps with the goal of performing a short proof of concept DNA synthesis using the lead TdT variant(s) and 3′-O- phosphate-nucleotides. Knowledge gained from Phase I protein engineering and short synthesis tests will guide further TdT improvements in Phase II towards synthesis of DNA with longer lengths and with lower error rates. The fully enzymatic synthesis cycle proposed to be developed represents a complete workflow for DNA synthesis, with commercial potential for implementation as a replacement for chemical DNA manufacturing.Project Narrative Access to long, accurate DNA sequences is a foundational technology for the field of synthetic biology, enabling access to novel therapeutics, biofuels, chemicals, and materials. Routine methods for DNA synthesis employ chemicals, which limits the lengths of the DNA that can be prepared and produces hazardous waste. Herein we propose development of a fully enzymatic synthesis cycle, which allows for a sustainable aqueous format and which provides a potential means for longer, more accurate DNA preparation.