SBIR Phase II: Reagent Development for a Rapid Enzymatic DNA Synthesis Platform

SBIR 第二阶段：快速酶促 DNA 合成平台的试剂开发

• 批准号: 2036532
• 负责人: Daniel Arlow
• 金额: $ 99.47万
• 依托单位: Ansa Biotechnologies, Inc.
• 依托单位国家: 美国
• 项目类别: Cooperative Agreement
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-15 至 2023-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2036532&HistoricalAwards=false
• 关键词: SBIR; Phase; II; Reagent; Development

The broader impact of this Small Business Innovation Research (SBIR) Phase II project is to optimize a system to rapidly synthesize long, user-defined sequences of DNA as a commercial service. The process will be capable of producing error-free DNA fragments, each long enough to encode multiple genes, in 1-2 weeks, at a price that will be affordable even to university research labs. Researchers currently can purchase short DNA fragments that must be stitched together to make functional genes – which is labor-intensive and sometimes impossible – or pay vendors to do it. The ability to buy an intact group of genes on one piece of DNA will help deepen the understanding of all biological systems, from animals and plants to the bacteria and viruses that infect them. Easy access to long DNAs will also allow synthetic biologists to build completely novel biological devices, such as bacteria that manufacture vitamins or medicines, cells that detect and destroy cancer, or new sustainable food ingredients and novel biomaterials.The proposed project is focused on developing an enzymatic method for DNA synthesis that will alleviate several problems inherent to chemical DNA synthesis, the only method currently available commercially. Chemical synthesis works well for short DNA fragments, but high-quality synthesis is limited to 200 bases. Longer fragments often are created by stitching together many short fragments, but this process is unreliable for sequences that contain repeats or high AT or GC content. In the proposed enzymatic DNA synthesis method, a single deoxynucleoside triphosphate (dNTP) is conjugated to the template-independent polymerase Terminal Deoxynucleotidyl Transferase (TdT) by a cleavable linker. When presented with a DNA primer, the dNTP-TdT conjugate adds its tethered nucleotide and remains attached, blocking further elongation by other conjugates. Cleavage of the linker releases the TdT, to be washed away with unreacted conjugates, and exposes the oligo for extension with the next dNTP-TdT conjugate. These two steps of “extension” and “deprotection” are iterated to synthesize a defined sequence. The accuracy of the resulting DNA strand depends on the stability and cleavage efficiency of the linker. A variety of linkages and cleavage enzymes will be tested to yield 99.9% stability and cleavage in rapid, 30-second reactions.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

小型企业创新研究(SBIR)第二阶段项目的更广泛影响是优化系统，以快速合成用户定义的长DNA序列作为商业服务。这一过程将能够在1-2周内生产出无错误的DNA片段，每个片段的长度足以编码多个基因，价格即使是大学研究实验室也能负担得起。研究人员目前可以购买短DNA片段，这些DNA片段必须缝合在一起才能形成功能基因--这是劳动密集型的，有时甚至是不可能的--或者付钱给供应商来做这件事。购买一段DNA上的一组完整基因的能力将有助于加深对所有生物系统的理解，从动植物到感染它们的细菌和病毒。容易获得长DNA还将使合成生物学家能够构建全新的生物设备，如制造维生素或药物的细菌、检测和摧毁癌症的细胞，或者新的可持续食品成分和新的生物材料。拟议的项目专注于开发一种用于DNA合成的酶方法，将缓解化学DNA合成固有的几个问题，这是目前唯一可用的商业方法。化学合成对短DNA片段很有效，但高质量的合成受200个碱基的限制。更长的片段通常是通过将许多短片段拼接在一起而产生的，但对于包含重复序列或高AT或GC含量的序列来说，这种过程是不可靠的。在所提出的酶促DNA合成方法中，单个脱氧核苷三磷酸(DNTP)通过可切割的连接子连接到模板非依赖的聚合酶末端脱氧核苷酸转移酶(TDT)上。当提供DNA引物时，dNTP-TDT连接物添加其拴系的核苷酸并保持连接，阻止其他连接物的进一步延伸。连接子的切割释放TDT，被未反应的连接物洗去，并暴露寡聚以与下一个dNTP-TDT连接物延伸。重复“扩展”和“解除保护”这两个步骤以合成定义的序列。所得DNA链的准确性取决于连接物的稳定性和切割效率。各种连接和裂解酶将进行测试，在30秒的快速反应中产生99.9%的稳定性和裂解。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。