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

这个小型企业创新研究(SBIR)项目的更广泛的影响/商业潜力是推动快速准确地合成DNA的新方法的开发。合成“基因长度”DNA分子(500个碱基)的能力对于生物学和生物医学研究来说是无价的，是快速发展的合成生物学领域的一项关键使能技术。现有的产生DNA序列的方法依赖于有机化学，通常仅限于直接合成200个碱基分子，需要进一步组装成基因长度的产品。然而，所使用的组装工艺容易失败，并且不能与所有所需的序列兼容。该项目正在开发的技术将展示一种基于酶的新DNA合成方法的开发，与有机化学相比，这种方法有望直接合成基因长度的DNA分子。酶法合成DNA的另一个好处是，它是在水条件下使用无毒试剂进行的，而化学DNA合成使用有毒试剂，并产生危险废物。酶法DNA合成方法一旦完全开发出来，将成为支持快速DNA合成服务的核心技术，这将加速生命科学和合成生物学的研究。这项SBIR第一阶段项目建议为一种新的酶法DNA合成方法开发优化的“TDT-dNTP偶联”试剂。酶的精致专一性有望使直接合成比目前使用的行业标准化学DNA合成方法长得多的DNA结构成为可能，从而加快合成生物学的工作流程。TDT-dNTP结合物由“模板无关的聚合酶”TDT和可通过TDT结合到DNA分子中的三磷酸核苷底物组成。在将被拴住的核苷酸添加到DNA分子后，聚合酶仍然通过与添加的核苷酸的系链连接到DNA分子的末端，阻止其他结合分子进一步延伸DNA分子，从而确保分子只延伸一个核苷酸。然后可以切断连接以释放聚合酶，并暴露DNA分子以供后续延伸。以前的研究已经证明，基于光解连接子的TdT-dNTP偶联物可以用于DNA的从头合成。该项目建议开发基于连接物的新TDT-dNTP连接物，该连接物可以使用水解酶快速且特定地切割，最终实现完全酶促DNA合成方法的前景。该奖项反映了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。

The broader impact/commercial potential of this Small Business Innovation Research (SBIR) project is to advance the development of a new method for rapid and accurate DNA synthesis. The ability to synthesize "gene-length" DNA molecules (500 bases) is invaluable for biological and biomedical research and is a key enabling technology for the rapidly growing field of synthetic biology. Existing methods for producing DNA sequences rely on organic chemistry and are generally limited to the direct synthesis of 200 base molecules, necessitating further assembly into gene-length products. However, the assembly process used is failure-prone and not compatible with all desired sequences. The technology being developed in this project will demonstrate the development of a new DNA synthesis method based on enzymes, which, in contrast to organic chemistry, promises to enable the direct synthesis of gene-length DNA molecules. An additional benefit of enzymatic DNA synthesis is that it is performed in aqueous conditions using nontoxic reagents, whereas chemical DNA synthesis uses toxic reagents and produces hazardous waste. The enzymatic DNA synthesis method, once fully developed, will be the core technology supporting a rapid DNA synthesis service that will accelerate research in life sciences and synthetic biology.This SBIR Phase I project proposes to develop optimized "TdT-dNTP conjugate" reagents for a new enzymatic DNA synthesis method. The exquisite specificity of enzymes promises to enable the direct synthesis of much longer DNA constructs than is possible with the industry-standard chemical DNA synthesis method in use today, accelerating workflows in synthetic biology. A TdT-dNTP conjugate consists of the "template-independent polymerase" enzyme TdT with a tethered nucleoside triphosphate substrate that can be incorporated into a DNA molecule by TdT. After adding the tethered nucleotide to a DNA molecule, the polymerase remains attached to the end of the DNA molecule via the tether to the added nucleotide, blocking other conjugate molecules from further extending the DNA molecule, and thus ensuring that the molecule only gets extended by one nucleotide. The linkage can then be cleaved to release the polymerase and expose the DNA molecule for subsequent extension. Previous research has demonstrated that TdT-dNTP conjugates based on a photocleavable linker can be used for stepwise de novo DNA synthesis. This project proposes to develop new TdT-dNTP conjugates based on a linker that can be rapidly and specifically cleaved using a hydrolase enzyme, finally realizing the promise of a fully-enzymatic DNA synthesis method.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.