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

基本信息

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
项目类别：
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

项目摘要

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，当前的DNA合成技术无法产生基因长度的DNA片段，并且依赖于昂贵且昂贵的技术。此外，目前的 DNA 合成方法是基于有机化学的，会产生难以处理且成本高昂的有毒废物混合物。在此，我们建议设计一种新型的基于生物学的 DNA 合成方法。启用高保真度、模板独立、长（> 500bp）DNA链的合成该提案描述了一种新颖的方法，该方法将降低每个合成生物学实验室的成本，从而实现更快的疫苗开发、生物分子计算、细胞重新编程等应用。该项目第二阶段开发的大规模并行合成能力将由分子组装公司用作定制合成服务，类似于当今定制寡核苷酸的订购、生产和交付方式。该提案的重点是改造酶末端脱氧核苷酸转移酶（TdT），该酶通过以不依赖模板的方式向单链 DNA 添加核苷酸来发挥作用，以利用修饰的脱氧核苷酸三磷酸（dNTP）。dNTP 类似物以这种方式被阻断。一次添加一种且仅一种选择的核苷酸，并且在添加到正在生长的链中之后，能够解封闭以再生天然DNA链。第一阶段涵盖工程酶的开发及其用于证明制造短序列特异性核酸的能力，第二阶段将引领这一阶段。对所有四种 dNTP 类似物、循环条件、自动化的优化以及这种新颖的聚脱氧核苷酸合成方法的全部功能的展示在 1981 年，很难想象 DNA 合成最终将在现代生物学中发挥的具体基本作用；而购买合成基因的想法只是科幻小说中的概念，人们只能想象按需、高纯度、低成本的多核苷酸将如何开启生物和临床应用的新时代。