Non-templated Enzymatic Synthesis of Polydeoxynucleotides

描述（由申请人提供）：在过去的30年里，用于常规应用的合成DNA的成本，如DNA测序，PCR和杂交分析，已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可能影响人类健康发展的许多生物应用的扩展。最后，目前的DNA合成方法是30年前开发的，以有机化学为基础，产生有毒的废物混合物，处理起来既困难又昂贵。该建议描述了一种新方法，它将降低每个分子生物学实验室的成本，并能够开发新的合成生物学应用，如更快地开发疫苗、生物分子计算、细胞重编程和改进细胞治疗。该项目的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，类似于今天由Molecular Assembly运营的定制寡核苷酸的订购、生产和交付方式。末端脱氧核苷酸转移酶的作用是以模板无关的方式在单链DNA上每次添加一个碱基。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，而不是产生类似长度的序列特异性多核苷酸。两个三磷酸脱氧核苷酸的方法描述了开发修改(核苷酸)类似物,以这样一种方式,是一种兼容TdT)和很容易添加到DNA链合成,b)阻塞的方式导致的一个且只有一个核苷酸的选择后,c)添加到链增长,能够de -阻塞,恢复自然的DNA链。第一阶段涵盖此类dNTP类似物的开发及其用于证明制造短序列特异性核酸的能力。第二阶段将优化所有四种dNTP类似物、循环条件、自动化，并展示这种新颖的多脱氧核苷酸合成方法的全部能力。如果是在1981年，情况就会如此

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.