Create Ultralong DNA Constructs in One Assembly Step

在一个组装步骤中创建超长 DNA 结构

• 批准号: 9754193
• 负责人: STEVEN A BENNER
• 金额: $ 64.84万
• 依托单位: FIREBIRD BIOMOLECULAR SCIENCES, LLC
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9754193
• 关键词: Agreement; Antibiotic Resistance; Architecture; Automation; Bacteriophages; Base Pairing; Basic Science; Benchmarking; Binding; Biotechnology; Businesses; C-glycoside; Cells; Chemicals; Collaborations; Computer software; Custom; DNA; DNA biosynthesis; Depurination; Ensure; Enzymes; Escherichia coli; Evaluation; Evaluation Research; Excision; Failure; Fluorescence; Foundations; Genes; Genetic; Genome; Ginkgo biloba; Human; Hydrogen Bonding; Information Systems; Investments; Kanamycin; Kanamycin Resistance; Legal patent; Length; Letters; Licensing; Marketing; Medical; Modeling; Molecular Evolution; Natural Products; Nucleotides; Occupations; Oligonucleotides; Outcome; Pattern; Periodicity; Phase; Plasmids; Privatization; Process; Production; Proteins; Reagent; Reproducibility; Residual state; Risk; Sales; Science; Secure; Services; Small Business Technology Transfer Research; Source; Speed; Technology; Technology Transfer; Therapeutic; United States National Institutes of Health; Update; Virus; base; commercialization; cost; cost shifting; deep sequencing; density; design; drug discovery; experimental study; gene synthesis; genetic information; improved; innovation; molecular recognition; nuclease; nucleobase; off-patent; phosphoramidite; prevent; product development; programs; self assembly; success; synthetic biology

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.

在一个组装步骤中创建超长DNA构建体 Firebird Biomolecular Sciences LLC Steven A. Benner 应用分子进化基金会 星香秀一 摘要 Frost & Sullivan发现，2014年全球DNA寡核苷酸市场为2.41亿美元，基因市场为1.37亿美元。私人 对Twist、Ginkgo和DNA Script等DNA合成公司的投资， 几十亿美元。国家卫生研究院、国防高级研究计划局和其他机构在“合成生物学”方面的联邦公共投资， 依赖于DNA合成每年超过1亿美元。这些数字支持这个项目， 两项创新（a）在定制合成模型下递送长DNA（L-DNA）组装体（B）确保了 许可平台，以及（c）创造合作和收购机会。这些技术是： 1.扩增的遗传信息系统（AEGIS），它添加4个核苷酸，形成2个额外的 将核碱基对（Z：P和S：B）与天然DNA中发现的对（C：G和T：A）正交结合。八- 字母DNA增加了可以自主组装的序列精确片段的数量。 2.音译，将Z，P，S和B分别转换为C，G，T和A，给出完全自然的L- AEGIS在完成组装片段的工作后，通过移除AEGIS组件来构建DNA。 第一阶段的主要成果包括： (a)OLIGARCHTM软件预测8-字母GACTZPSB DNA双链体的稳定性。 (b)AEGIS +音译法生产的DNA产品的保真度与商业G块一样好。 (c)构建的卡那霉素抗性基因和绿色荧光蛋白基因在大肠杆菌中具有活性。coli细胞。 这些成功将L-DNA合成的成本/质量负担转移到残差管理上。 目标1.如实验所示，使用以下方法管理残差： 1.1 C-糖苷，以消除脱嘌呤和脱嘧啶，如果这些引起残留误差。 1.2酶促可去除的保护基团，以消除脱保护期间的化学损伤。 1.3.可捕获的封端基团，以实现>99.999%的截短物质的简单去除。 1.4酶促DNA合成，消除“苛刻”亚磷酰胺合成中的所有“化学”降解。 将使用MutS和Surveyor核酸酶误差校正进一步管理残留误差。 目标2.创建合成管道，以准备用于管理残差的构建模块和试剂。 目标3.开发基于阵列的亚磷酰胺合成片段，并进行连续错误评估。 将通过使试剂本身可用于销售来确保重现性。这是一个来源， 直接收入以及我们的营销策略的主要部分。目前，试剂销售情况令人满意 客户已经产生了价值超过250万美元的许可协议。为了商业化，Firebird刚刚执行了一项 与非模板化酶促DNA合成及其自动化的先驱DNA Script达成协议， 在第2阶段之后，基于酶的DNA合成是否应该被优先用于管理残留误差。这 包括授权Firebird的酶循环可逆终止非模板DNA合成专利。