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.

项目概要/摘要 DNA 合成彻底改变了合成生物学领域，催生了新的生物疗法 合成 DNA 的化学方法已被开发了 30 多种。 几年前，仍然面临着高成本和 DNA 长度限制（<200 个核苷酸）的挑战。 生物学已经超越了当前的 DNA 合成技术，许多研究方法的范围现在已经扩大 受限于合成 DNA 的成本和长度。 聚合酶以及封闭核苷酸 (dNTP) 的逐步掺入和脱保护以及 尽管具有潜力，但它是克服化学 DNA 合成局限性的一种有前途的替代方案。 大多数酶促方法仍然依赖于化学处理步骤来去除封闭基团 化学解封闭步骤会产生危险废物并重复进行。 在此第一阶段 SBIR 提案中，Molecular Assemblies Inc. 建议开发一种全酶促 DNA 合成方法，该方法的核心是三个关键。 酶促步骤：1) 聚合酶掺入 3'-O-封闭核苷酸，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) 一种新颖的酶清理方法 通过利用酶而不是化学的效率和特异性来消耗未反应的物质。 过程中，我们寻求开发一种环境友好的 DNA 合成方法，目标是 生成更长（>200 个核苷酸）、更纯的 DNA 是这项工作的一个关键目标。 不依赖模板的聚合酶，末端脱氧核苷酸转移酶 (TdT)，用于改进 3′-O- 我们将使用 1) 合理设计氨基酸突变。 蛋白质设计软件 Rosetta，以及 2) 计算机生物勘探，以产生包含以下内容的筛选文库 这种组合酶工程方法具有丰富的系统发育多样性。 鉴定具有不同表型的酶突变体的潜力，我们将表达并筛选所得的结果。 使用我们建立的高通量核苷酸掺入分析来鉴定目标文库 然后，我们将使用最活跃的 TdT 变体优化酶清理和解封闭步骤。 使用主要 TdT 变体和 3′-O- 进行简短的概念 DNA 合成验证的目标 从第一阶段蛋白质工程和短合成测试中获得的知识。 将指导第二阶段进一步 TdT 改进，以合成更长长度的 DNA 拟开发的全酶合成循环代表了一个完整的错误率。 DNA 合成工作流程，具有替代化学物质的商业潜力 DNA制造。