Modified Nucleotidyl Transferases for Enzymatically Mediated Oligodeoxynucleotide Synthesis

 描述(申请人提供)：在快速发展的合成生物学领域，用于基因合成的合成DNA的成本已成为许多实验室预算的重要组成部分。目前的DNA合成技术无法产生基因长度的DNA片段，并且依赖昂贵且容易出错的组装方法来构建长链DNA。此外，目前的DNA合成方法是以有机化学为基础的，产生的有毒废物混合物难以处理，处理成本也很高。在这里，我们建议设计一种新的基于生物的DNA合成方法，该方法将能够高保真、不依赖模板地合成长(&gt；500bp)DNA链。这项提议描述了一种新的方法，它将导致每个合成生物学实验室的成本降低，使诸如更快的疫苗开发、生物分子计算、细胞的重新编程和改进的细胞疗法等应用成为可能。在该项目第二阶段开发的大规模并行合成能力将作为分子组装公司的定制合成服务使用，类似于目前定制低聚物的订购、生产和交付方式。为了实现这一目标，这项提议的重点是改造酶末端脱氧核苷酸转移酶(TDT)，它的作用是以模板无关的方式将核苷酸添加到单链DNA上，以利用修饰的脱氧核苷酸三磷酸(DNTPs)。DNTP类似物被阻止的方式导致一次添加一个且只有一个选择的核苷酸，并且在添加到生长链中后，能够被解除阻止以再生天然DNA链。第一阶段包括开发一种工程酶，并用它来证明制造短序列特定核酸的能力。第二阶段将主导 优化所有四个dNTP类似物、循环条件、自动化，并展示这种新的、合成多脱氧核苷酸的方法的全部能力。在1981年，很难想象DNA合成最终会在现代生物学中扮演什么特定的基础角色；而购买合成基因的想法还只是科幻小说中的一个概念。人们只能想象按需、高纯度、低成本的多核苷酸将如何使生物和临床应用进入一个新时代。

 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.