Modified Nucleotidyl Transferases for Enzymatically Mediated Oligodeoxynucleotide Synthesis

用于酶介导的寡脱氧核苷酸合成的修饰核苷酸转移酶

• 批准号: 8904404
• 负责人: John W Efcavitch
• 金额: $ 18.9万
• 依托单位: MOLECULAR ASSEMBLIES, INC.
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8904404
• 关键词: Active Sites; Aliquot; Amino Acids; Automation; Binding; Biological; Biological Assay; Biology; Budgets; Buffers; Cells; Collaborations; Computing Methodologies; Custom; DNA; DNA Nucleotidylexotransferase; DNA Sequence; DNA biosynthesis; DNA chemical synthesis; Development; Engineering; Enzymes; Escherichia coli; Failure; Gel; Genes; Genomics; Goals; Health; Health Services Research; Hour; Human; Ions; Laboratories; Lead; Length; Libraries; Link; Manuals; Mediating; Methods; Modeling; Modification; Molecular Biology; Molecular Models; Monitor; Mutation; N-terminal; Natural regeneration; Nucleic Acids; Nucleotides; Oligonucleotides; Organic Chemistry; Phase; Plant Resins; Play; Polymerase; Polynucleotides; Positioning Attribute; Process; Proteins; Reaction; Reagent; Recombinants; Role; Science; Services; Single-Stranded DNA; Solid; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spottings; Synthetic Genes; Technology; Therapeutic; Time; Transferase; Transferase Gene; Validation; Variant; analog; aqueous; base; clinical application; combinatorial; cost; design; enzyme activity; expression vector; fictional works; gene synthesis; improved; model design; molecular assembly/self assembly; molecular modeling; mutant; novel; nucleotide analog; phosphoramidite; prototype; public health relevance; screening; synthetic biology; synthetic construct; tripolyphosphate; vaccine development; wasting

 DESCRIPTION (provided by applicant): In the rapidly growing field of synthetic biology the cost of synthetic DNA for gene synthesis has become a substantial part of many laboratory budgets. Current DNA synthesis technologies are unable to produce gene length DNA fragments and rely on expensive and error prone assembly methods to construct long strands of DNA. In addition, current DNA synthesis methods are organic chemistry based and produce toxic waste mixtures that are difficult and costly to dispose of. Here we propose to design a novel biologically based DNA synthesis method which will enable the high fidelity, template independent, synthesis of long (>500bp) strands of DNA. This proposal describes a novel method that will lead to reduced costs in every synthetic biology laboratory, enabling applications such as faster development of vaccines, biomolecular computation, reprograming of cells and improved cellular therapeutics. The resulting massively parallel synthesis capability developed in Phase II of this project will be put to use as a custom synthesis service by Molecular Assemblies similar to how custom oligos are ordered, produced and delivered today. To achieve this, this proposal focuses on engineering the enzyme terminal deoxynucleotidyl transferase (TdT), which acts by adding nucleotides to single stranded DNA in a template-independent fashion, to utilize modified deoxynucleotide triphosphates (dNTPs). The dNTP analogs are blocked in such a way that leads to the addition of one and only one nucleotide of choice at a time and, after being added to the growing strand, are able to be de-blocked to regenerate a natural DNA strand. Phase I covers the development of an engineered enzyme and its use to prove the ability to make a short sequence specific nucleic acid. Phase II will lead to optimization of all four dNTP analogs, cycle conditions, automation and the demonstration of the full capabilities of this novel, synthetic approach for polydeoxynucleotides. In 1981 it would have been difficult to envision the specific fundamental roles DNA synthesis would eventually play in modern biology; while the idea of purchasing synthetic genes was a concept of science fiction. One can only imagine how on-demand, high purity, low cost polynucleotides will enable a new era of biological & clinical applications.

 描述（由适用提供）：在合成生物学的快速增长领域中，基因合成的合成DNA成本已成为许多实验室预算的第二部分。当前的DNA合成技术无法产生基因长度DNA片段，并且依赖于昂贵且易于误解的组装方法来构建DNA的长链。另外，当前的DNA合成方法是基于有机化的，并且产生了困难且昂贵的有毒废物混合物。在这里，我们建议设计一种新型的基于生物学的DNA合成方法，该方法将使DNA的长（> 500bp）链的高忠诚度，模板独立，合成。该提案描述了一种新的方法，该方法将导致每个合成生物学实验室的成本降低，从而使应用能够更快地开发疫苗，生物分子计算，重新编程，细胞重编程和改善的细胞疗法。在该项目的第二阶段中开发的大规模平行合成能力将通过分子组件用作自定义合成服务，类似于今天订购，生产和交付自定义寡聚的方式。为了实现这一目标，该提案着重于工程酶末端脱氧核苷酸转移酶（TDT），该酶通过以模板独立的方式将核苷酸添加到单个绞线DNA中，以利用修饰的脱氧核苷酸脱氧核苷酸triphotide triphothates（DNTPS）。 DNTP类似物以这样的方式被阻断，从而导致一次添加一个和仅选择的核苷酸，并且在添加到生长的链中后，能够被去除以再生自然DNA链。第一阶段涵盖了工程酶的开发及其用来证明制作短序列特异性核酸的能力。第二阶段将领导 为了优化所有四个DNTP类似物，周期条件，自动化以及这种新颖的多脱氧核苷酸合成方法的完整能力。 1981年，很难想象DNA综合最终将在现代生物学中发挥的特定基本作用。购买合成基因的想法是科幻小说的概念。人们只能想像一下，按需，高纯度，低成本的多核苷酸将如何实现生物学和临床应用的新时代。