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

项目摘要/摘要 DNA合成使合成生物学领域发生了革命性的变化，导致了新的治疗方法，基于生物的 燃料、化学品和材料。合成DNA的化学方法是在30多年前发展起来的 几年前，仍然受到高昂的成本和DNA长度(200个核苷酸)限制的挑战。作为合成材料 生物学已经超过了目前的DNA合成技术，许多研究方法的范围现在 受合成DNA的成本和长度的限制。利用酶促合成DNA的方法 聚合酶与阻断核苷酸(DNTPs)的逐步掺入和去保护 是克服化学DNA合成局限性的一种很有前途的替代方法。尽管他们有潜力， 大多数酶处理方法仍然依赖于化学处理步骤来去除封闭基团 合成序列。化学解堵步骤会产生危险废物，并反复受到 将寡核苷酸转化为可降解的化学物质。在这个第一阶段的SBIR提案中，分子组装公司。 建议开发一种完全酶促的DNA合成方法。这种方法的核心有三个关键 酶促步骤：1)聚合酶掺入3‘-O-封闭的核苷酸，2)酶解封闭 从3‘-羟基上去除磷酸盐封闭基团的步骤，以及3)一种新的酶促清理，以 耗尽未反应的物质。通过利用酶而不是化学物质的效率和特异性 过程，我们寻求开发一种环境友好的DNA合成方法，目标是 产生更长的(&gt；200个核苷酸)，更纯的DNA。拟议工作的一个关键目标是设计 模板非依赖聚合酶，末端脱氧核苷酸转移酶(TDT)，用于改进的3‘-O- 磷酸dNTP掺入。我们将结合1)合理设计的氨基酸突变使用 蛋白质设计软件Rosetta，和2)在硅胶生物勘探中产生筛选文库，包括 系统发育不同的TDT背景。这种结合酶工程的方法具有很好的 鉴定具有不同表型的酶突变体的可能性。我们将表达和筛选由此产生的 使用我们建立的高通量核苷酸掺入试验来鉴定靶向文库 最活跃的TDT变种。然后，我们将优化酶清理和解堵步骤 目标是使用领先的TDT变异体(S)和3‘-O-进行简短的概念验证DNA合成 磷酸-核苷酸。从第一阶段蛋白质工程和短合成试验中获得的知识 将在第二阶段指导TDT的进一步改进，以合成更长长度的DNA和 更低的错误率。建议开发的完全酶促合成循环代表着一个完整的 DNA合成工作流程，具有作为化学品替代品的商业潜力 DNA制造。

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 (<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 (>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.