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

这个小型企业创新研究（SBIR）第二阶段项目的更广泛的影响是优化一个系统，以快速合成长的，用户定义的DNA序列作为商业服务。该过程将能够在1-2周内产生无错误的DNA片段，每个片段的长度足以编码多个基因，其价格甚至可以让大学研究实验室负担得起。目前，研究人员可以购买短的DNA片段，这些片段必须拼接在一起才能产生功能基因--这是劳动密集型的，有时是不可能的--或者付钱给供应商。购买一段DNA上的完整基因组的能力将有助于加深对所有生物系统的理解，从动物和植物到感染它们的细菌和病毒。合成生物学家也可以轻松获得长DNA，从而构建全新的生物设备，例如制造维生素或药物的细菌、检测和破坏癌症的细胞、新的可持续食品成分和新的生物材料。该项目的重点是开发一种用于DNA合成的酶法，该方法将减轻化学DNA合成所固有的几个问题，化学DNA合成是目前唯一的商业方法。化学合成对于短的DNA片段效果很好，但高质量的合成限于200个碱基。较长的片段通常是通过将许多短片段拼接在一起而产生的，但对于含有重复序列或高AT或GC含量的序列，该过程是不可靠的。在所提出的酶促DNA合成方法中，单个脱氧核苷三磷酸（dNTP）通过可切割接头与模板非依赖性聚合酶末端脱氧核苷酸转移酶（TdT）缀合。当与DNA引物一起呈现时，dNTP-TdT缀合物添加其拴系的核苷酸并保持连接，从而阻止其他缀合物的进一步延伸。接头的切割释放TdT，其将与未反应的缀合物一起被洗掉，并暴露寡核苷酸用于与下一个dNTP-TdT缀合物一起延伸。重复“延伸”和“脱保护”这两个步骤以合成限定的序列。所得DNA链的准确性取决于接头的稳定性和切割效率。各种连接和裂解酶将被测试，以产生99.9%的稳定性和裂解在快速，30秒的reactions.This奖项反映了NSF的法定使命，并已被认为是值得的支持，通过评估使用基金会的知识价值和更广泛的影响审查标准。

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.