Non-templated Enzymatic Synthesis of Polydeoxynucleotides

描述(由申请人提供)：在过去的30年里，用于DNA测序、聚合酶链式反应和杂交分析等常规应用的合成DNA的成本已经成为许多实验室预算的重要组成部分。此外，长度和质量问题限制了许多生物应用的扩展，这可能会影响人类健康的发展。最后，目前30年前开发的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。这项提议描述了一种新的方法，它将降低每个分子生物学实验室的成本，并使新的合成生物学应用的开发成为可能，例如更快地开发疫苗、生物分子计算、细胞的重新编程和改进的细胞疗法。该项目第二阶段开发的大规模并行合成能力将作为定制合成服务使用，类似于目前由分子组装公司运营的定制寡聚的订购、生产和交付方式。酶末端脱氧核苷酸转移酶的作用是以模板无关的方式一次向单链DNA添加一个碱基。该项目的目标是利用TDT的显著特性，它将聚合数千个核苷酸长的均聚物，从而产生类似长度的序列特异性多核苷酸。该方法描述了两个脱氧核糖核苷酸三磷酸(DNTP)类似物的发展，它们以这样的方式被修饰，使得它们a)与TDT相容并且容易地添加到正在合成的DNA链中，b)以导致一次仅添加一个选择的核苷酸的方式被阻止，以及c)在被添加到生长链中之后，能够以能够以再生天然DNA链的方式去阻止。第一阶段包括开发这种dNTP类似物，并用它们来证明制造短序列特异性核酸的能力。第二阶段将对所有四个dNTP类似物、循环条件、自动化进行优化，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，它会有 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本角色；人类基因组测序的引子、聚合酶链式反应和杂交阵列。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): Over the last 3 decades the cost of synthetic DNA for routine applications such as DNA sequencing, PCR and hybridization analysis has become a substantial part of many laboratory budgets. Additionally, length and quality issues limit the expansion of numerous biological applications which could impact human health developments. Finally, current DNA synthesis methods, developed 30 years ago are organic chemistry based and produce toxic waste mixtures that are difficult and costly to dispose of. This proposal describes a novel method that will lead to reduced costs in every molecular biology laboratory and enable the development of new synthetic biology 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, similar to how custom oligos are ordered, produced and delivered today, run by Molecular Assembly. The enzyme terminal deoxynucleotidyl transferase acts by adding one base at a time to a single stranded DNA in a template-independent fashion. The goal of this project is to harness the remarkable properties of TdT which will polymerize homopolymers thousands of nucleotides long, to instead produce sequence specific polynucleotides of similar lengths. The approach describes development of two deoxyribonucleotide triphosphate (dNTP) analogs that are modified in such a way so that they are a) compatible with TdT and are readily added to a strand of DNA being synthesized, b) blocked in such a way that leads to the addition of one and only one nucleotide of choice at a time and c) after being added to the growing strand, able to be de- blocked in such a way that regenerates a natural DNA strand. Phase I covers the development of such dNTP analogs and their use to prove the ability to make a short sequence specific nucleic acid. Phase II will lead t 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; primers for sequencing the human genome, PCR and hybridization arrays. One can only imagine how on-demand, high purity, low cost polynucleotides will enable a new era of biological & clinical applications.