Create Ultralong DNA Constructs in One Assembly Step

在一个组装步骤中创建超长DNA构建体 火鸟生物分子科学有限责任公司 史蒂文·A·本纳 应用分子进化基础 星间水一 摘要 Frost&Sullivan发现，2014年DNA寡核苷酸的全球市场价值为2.41亿美元，基因市场为1.37亿美元。私 对Twist、Ginkgo和DNA Scrip等DNA合成公司的投资给出了 数十亿美元。美国国家卫生研究院、美国国防部高级研究计划局和其他机构在“合成生物学”领域的联邦公共投资 依赖于DNA合成每年超过1亿美元。这些数字支持着这个项目的发展 两项创新：(A)在定制合成模型下，提供长DNA(L-DNA)组件(B)确保 许可平台，以及(C)创造协作和买断机会。这些技术包括： 1.人工扩展遗传信息系统(Aegis)，其添加4个核苷酸形成2个额外的 将碱基对(Z：P和S：B)与天然脱氧核糖核酸中的碱基对(C：G和T：A)正交结合。八个人- 字母DNA增加了可以自主组装的序列准确片段的数量。 2.音译，将Z、P、S、B分别转换为C、G、T、A，给出一个完全自然的L-- 在宙斯盾组件完成组装碎片的工作后，移除宙斯盾组件来构建DNA。 第一阶段成果的亮点包括： (A)OLIGARCHTM软件预测8字母GACTZPSB DNA双链的稳定性。 (B)通过宙斯盾+音译制作的DNA产品的保真度与商业G形块一样好。 (C)构建的卡那霉素抗性基因和绿色荧光蛋白在大肠杆菌中具有活性。 这些成功将L DNA合成的成本/质量负担转移到了残差管理上。 目标1.如实验所示，使用以下工具管理残差： 1.1 C-糖苷，以消除脱嘌呤和脱嘧啶，如果这些造成残留误差。 1.2酶可移除保护基团，以消除解除保护过程中的化学损害。 1.3.可捕获封顶组，实现简单&gt；99.999%的截短物种去除。 1.4酶促脱氧核糖核酸(DNA)合成，以消除“苛刻”亚磷酰胺合成中的所有“化学”降解。 残余误差将使用MutS和Surveyor核酸酶误差校正进行进一步管理。 目标2.创建合成管道，以准备用于管理残余误差的积木和试剂。 目的3.发展基于阵列的亚磷酰胺片段合成，并进行连续误差评估。 通过使试剂本身可供销售，将确保重复性。这是一个来源 即时收入也是我们营销战略的重要组成部分。试剂销售已经令人满意 客户已经达成了价值超过250万美元的许可协议，用于商业化，Firebird刚刚签署了一份 与DNA脚本达成协议，DNA脚本是非模板化酶法DNA合成及其自动化的先驱，To Go 在第二阶段之后，是否应该首选以酶为基础的DNA合成来管理残留误差。这 包括许可火鸟公司用于酶环可逆终止非模板DNA合成的专利。

Create Ultralong DNA Constructs in One Assembly Step Firebird Biomolecular Sciences LLC Steven A. Benner Foundation for Applied Molecular Evolution Shuichi Hoshika Abstract Frost & Sullivan found a 2014 global market for DNA oligos at $241 million, $137 million for genes. Private investment in DNA synthesis companies like Twist, Ginkgo, and DNA Script give collective valuations of several billion dollars. Federal public investment by the NIH, DARPA, and others in "synthetic biology" that depends on DNA synthesis exceeds $100 million annually. These numbers stand behind this project to develop two innovations to (a) deliver, under a custom synthesis model, long DNA (L-DNA) assemblies (b) secure a licensing platform, and (c) create collaboration and buyout opportunities. These technologies are: 1. Artificially expanded genetic information systems (AEGIS), which add 4 nucleotides forming 2 additional orthogonally binding nucleobase pairs (Z:P and S:B) to the pairs (C:G and T:A) found in natural DNA. Eight- letter DNA increases the number of sequence accurate fragments that can be autonomously assembled. 2. Transliteration, which converts Z, P, S and B to C, G, T and A respectively, giving an entirely natural L- DNA construct by removing the AEGIS components after they have done their job assembling fragments. Highlights of Phase I results include: (a) OLIGARCHTM software predicting stability of 8-letter GACTZPSB DNA duplexes. (b) Fidelity of DNA products made by AEGIS + transliteration as good as in commercial G-blocks. (c) Constructed genes for kanamycin resistance and green fluorescence protein were active in E. coli cells. These successes shift the cost/quality burden for L-DNA synthesis towards residual error management. Aim 1. Manage residual error using, as experiments suggest: 1.1 C-glycosides to eliminate depurination and depyrimidinylation, should these cause residual error. 1.2 Enzymatically removable protecting groups to eliminate chemical damage during deprotection. 1.3. Capturable capping groups to achieve simple >99.999% removal of truncated species. 1.4 Enzymatic DNA synthesis to eliminate all "chemical" degradation in "harsh" phosphoramidite synthesis. Residual error will be further managed using MutS and Surveyor nuclease error correction. Aim 2. Create synthetic pipelines to prepare the building blocks and reagents used to manage residual error. Aim 3. Develop array-based phosphoramidite synthesis of fragments with continuous error evaluation. Reproducibility will be ensured by making the reagents themselves available for sale. This is a source of immediate revenue as well as a major part of our marketing strategy. Already, reagent sales to satisfied customers have yielded licensing deals worth over $2.5 MM. For commercialization, Firebird just executed an agreement with DNA Script, a pioneer for non-templated enzymatic DNA synthesis and its automation, to go forward after Phase 2, should enzyme-based DNA synthesis be preferred to manage residual error. This includes licensing Firebird's patents for enzymatic cyclic reversibly terminated untemplated DNA synthesis.