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

通过工程模板独立聚合酶和新型磷酸 dNTP 封闭基团开发全酶促寡核苷酸合成循环

• 批准号: 10201535
• 负责人: Natasha Paul
• 金额: $ 25.62万
• 依托单位: MOLECULAR ASSEMBLIES, INC.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-16 至 2022-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10201535
• 关键词: Active Sites; Algorithm Design; Alkaline Phosphatase; Amino Acids; Biological Assay; Blood capillaries; Charge; Chemicals; Chemistry; Computer software; DNA; DNA Damage; DNA Nucleotidylexotransferase; DNA Sequence; DNA biosynthesis; DNA chemical synthesis; Development; Engineering; Enzymes; Excision; Exhibits; Exodeoxyribonuclease I; Family member; Formulation; Foundations; Goals; Hazardous Waste; Homologous Gene; Hydroxyl Radical; Individual; Knowledge; Lead; Length; Libraries; Mass Spectrum Analysis; Measurement; Methods; Modeling; Molecular Biology; Mutagenesis; Mutation; Nucleotides; Oligonucleotide Primers; Oligonucleotides; Phase; Phenotype; Phylogenetic Analysis; Polymerase; Preparation; Primer Extension; Process; Protein Engineering; Proteins; Protocols documentation; Research; Screening Result; Shapes; Shrimp; Site; Small Business Innovation Research Grant; Software Design; Source; Specificity; Technology; Testing; Treatment Step; Variant; Work; analog; aqueous; base; cost; design; experimental study; gel electrophoresis; genome editing; high throughput screening; improved; in silico; inorganic phosphate; molecular assembly/self assembly; mutant; next generation sequencing; novel; novel therapeutics; nucleotide analog; phosphoramidite; screening; synthetic biology; wasting

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.

项目总结/摘要 DNA合成已经彻底改变了合成生物学领域，导致新的治疗方法，生物基 燃料、化学品和材料。合成DNA的化学方法发展了30多年， 多年前，它仍然受到高成本和DNA长度限制（<200个核苷酸）的挑战。诸如合成 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是 受到成本和合成DNA长度的限制。酶促DNA合成方法采用 聚合酶和阻断核苷酸（dNTP）的逐步掺入和脱保护， 是克服化学DNA合成局限性的一种有前途的替代方法。尽管他们有潜力， 大多数酶促方法仍然依赖于化学处理步骤以从酶解产物中除去封闭基团。 合成序列化学解封步骤会产生危险废物，并重复进行 寡核苷酸降解化学品。在第一阶段SBIR提案中，Molecular Assemblies Inc. 提出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）酶促去封闭 从3′-羟基上除去磷酸封闭基团的步骤，和3）一种新的酶清除， 耗尽未反应的材料。通过利用酶的效率和特异性，而不是化学 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（>200个核苷酸）、更纯的DNA。拟议工作的一个主要目标是设计 非模板依赖性聚合酶，末端脱氧核苷酸转移酶（TdT），用于改进3′-O- 磷酸dNTP掺入。我们将结合1）使用氨基酸突变的合理设计， 蛋白质设计软件Rosetta，和2）计算机生物勘探，以产生筛选文库，包括 遗传学上不同的TdT背景。这种组合的酶工程方法具有很大的 有可能鉴定具有不同表型的酶突变体。我们将表达和筛选结果 靶向文库使用我们建立的高通量核苷酸掺入测定来鉴定 最活跃的TdT变体。然后，我们将优化酶清除和去封闭步骤， 目标是使用前导TdT变体和3′-O- 磷酸核苷酸。从I期蛋白质工程和短合成试验中获得的知识 将指导TdT在第二阶段的进一步改进，以合成更长长度的DNA， 更低的错误率。建议开发的完全酶促合成循环代表了一个完整的 DNA合成的工作流程，具有替代化学合成的商业潜力， DNA制造