Synthesis of Modified Nucleotide Triphosphates for Enzymatic DNA Synthesis**

用于酶促 DNA 合成的修饰核苷酸三磷酸的合成**

• 批准号: 536353-2018
• 负责人: Kraatz, HeinzBernhard
• 金额: $ 1.82万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Engage Grants Program
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=655959
• 关键词: Synthesis; Modified; Nucleotide; Triphosphates; Enzymatic

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.

DNA测序技术使阅读DNA的价格在10年内下降了10,000多倍。合成生物学的瓶颈现在是编写（即合成）DNA。三个主要经济体（美国、法国、英国）的初创公司正在研究下一代酶促DNA合成技术，以取代目前的化学技术。Spindle Biotech专注于开发加拿大酶促DNA合成技术。目前，唯一商业上可行的方法是20世纪70年代开发的两步法。这种方法化学合成短的DNA片段，并在一个容易出错和耗时的步骤中组装它们。这一过程所需时间要长一个数量级，估计每年产生1，200，000升危险化学废物，包括乙腈、三氯乙酸、甲苯、四氢呋喃和吡啶。酶促DNA合成是降低长合成DNA的成本和周转时间的唯一方法，这有助于加速疫苗生产，设计能够限制癌症生长的新型酶，生产可持续的生物燃料，提高作物产量，以及检测和分解土壤，空气和水中的环境污染物。所提出的创新是一组十六种受保护的脱氧核糖核苷酸三磷酸，对应于具有选自文献的四个保护基团的四种天然核苷酸的每个排列。核苷酸将在斯卡伯勒的多伦多大学合成。核苷酸将由Spindle Biotech Inc.筛选。在多伦多市中心的JLABS孵化器。针对核苷酸文库的酶和反应条件来优化DNA合成过程。已经确定了拟议综合的一些出发点。工业合作伙伴Spindle Biotech Inc.是一家加拿大独资公司，专注于开发加拿大酶促DNA合成技术。在这个项目中设计的核苷酸将被纳入一个DNA合成器正在平行开发。影响深远，包括公共卫生和国家安全。