Synthesis of Modified Nucleotide Triphosphates for Enzymatic DNA Synthesis**

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DNA sequencing technology has dropped the price of reading DNA over 10,000 times in 10 years. The bottleneck of synthetic biology is now writing (i.e. synthesizing) DNA. Startups in three major economies (US, France, UK) are working on next-generation enzymatic DNA synthesis technology to replace the current chemical technology. Spindle Biotech is solely focused on developing Canadian technology for enzymatic DNA synthesis. Currently, the only commercially viable method is a two-step process developed in the 1970s. This method chemically synthesizes short DNA fragments and assembles them in an error-prone and time-consuming step. This process takes an order of magnitude longer and is responsible for an estimated 1,200,000 litres of hazardous chemical waste per year, including acetonitrile, trichloroacetic acid, toluene, tetrahydrofuran, and pyridine. Enzymatic DNA synthesis is the only way to reduce the cost and turnaround time for long synthetic DNA, which helps in accelerating vaccine production, engineering novel enzymes that have the ability to restrict cancer growth, producing sustainable biofuels, improving crop yield, and detecting and breaking down environmental pollutants in the soil, air, and water.**The proposed innovation is a set of sixteen protected deoxyribonucleotide triphosphates, corresponding to the each permutation of the four natural nucleotides with four protecting groups chosen from literature. The nucleotides will be synthesized at the University of Toronto, Scarborough. The nucleotides will be screened by Spindle Biotech Inc. at the JLABS incubator in downtown Toronto. Nucleotides against a library of enzymes and reaction conditions to optimize the DNA synthesis process. A number of starting points for the proposed syntheses have been identified. The industrial partner, Spindle Biotech Inc., is a Canadian-owned company solely focused on developing Canadian technology for enzymatic DNA synthesis. The nucleotides designed in this project will be incorporated into a DNA synthesizer being developed in parallel. The implications are far reaching, including public health and national security.