CAS: Long Oligodeoxynucleotides Directly from Automated De Novo Synthesis

有了这个奖项，化学系的化学合成计划正在支持密歇根理工大学化学系的方世跃博士的研究，以开发用于制备和纯化长寡脱氧核苷酸（ODNs）的自动化工具。长ODN是化学制备的DNA（2 '-脱氧核糖核酸）分子，包含60个或更多个称为核苷酸的单元。它们用于基因组构建、疫苗开发和降低DNA数字存储数据的投入成本。目前，超过200个核苷酸的ODN的制备是繁琐的，并且需要低效地制备和纯化许多短ODN，然后进行分子生物学连接技术。方博士和他的团队正在努力解决这一瓶颈，以推进需要这些化合物的合成生物学研究。正在开发新的可持续技术，以可靠地从复杂的反应混合物中制备和分离具有500个或更多核苷酸的长ODN，而不会产生大量的溶剂废物。Yinan Yuan博士也在MTU，为ODN表征提供专家帮助。该项目正在为MTU的研究生和本科生以及附近主要为本科生的芬兰大学的本科生提供跨学科培训机会。开发制备长ODN的有效方法对于合成生物学的进步、核酸疫苗的开发和DNA数字存储数据至关重要。由于各种障碍，现有技术不能在这些研究领域的低成本和短周转时间要求内生产长ODN。方教授及其团队正在采取整体方法来实现长ODN合成的有效方法，并通过以下活动来解决这一重要需求：1）确定长ODN合成的最佳固体载体; 2）发明新的亚磷酰胺以提高富含dG区域的偶联产率; 3）研究用于长ODN纯化的聚合捕获技术; 4）研究用于长ODN纯化的聚合捕获技术。4）开发基于MALDI-MS（基质辅助激光解吸电离-质谱）的长ODN的改进表征方法;和5）设计用于长ODN中错误的酶促校正的方案。通过这些目标，方博士和袁博士正在朝着从头500-mer ODN合成的常规自动化方向取得进展。 成功的关键是使用聚合捕获技术来消除失败序列并简化纯化。朝着这些目标努力的研究生和本科生学习广泛的技能，包括有机合成，核酸化学，表面反应，自动合成，MALDI-MS，电泳和DNA操作和测序。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

With this award, the Chemical Synthesis Program of the Division of Chemistry is supporting the research of Dr. Shiyue Fang in the Department of Chemistry at Michigan Technological University to develop automated tools for making and purifying long oligodeoxynucleotides (ODNs). Long ODNs are chemically prepared DNA (2'-deoxyribonucleic acid) molecules that contain 60 or more units called nucleotides. They are used for genome construction, vaccine development, and reducing input costs of DNA digital storage data. Currently, the preparation of ODNs longer than 200 nucleotides is tedious and requires the inefficient preparation and purification of many short ODNs followed by molecular biology linking techniques. Dr. Fang and his team are working to address this bottleneck to advance synthetic biology studies that require these compounds. New sustainable technology is being developed to reliably make and separate long ODNs with 500 or more nucleotides from complex reaction mixtures without generating large amounts of solvent waste. Dr. Yinan Yuan, also at MTU, is contributing expert assistance with ODN characterization. The project is providing interdisciplinary training opportunities for a diverse group of graduate and undergraduate students at MTU, as well as undergraduate students from Finlandia University, a nearby primarily undergraduate institution. The development of efficient methods for preparing long ODNs is critically important for the advancement of synthetic biology, nucleic acid vaccine development, and DNA digital storage data. Due to a variety of roadblocks, existing technologies cannot produce long ODNs within the low cost and short turnaround time demands of these research fields. Professor Fang and his team are taking a holistic approach to achieving efficient methods for long ODN synthesis and addressing this important need through the following activities: 1) identifying optimal solid supports for long ODN synthesis; 2) inventing new phosphoramidites to improve coupling yields in dG-rich regions; 3) investigating catching-by-polymerization technology for long ODN purification; 4) developing improved characterization methods for long ODNs based on MALDI-MS (matrix-assisted laser desorption ionization-mass spectrometry); and 5) designing protocols for enzymatic correction of errors in long ODNs. Through these objectives, Drs. Fang and Yuan, are making progress toward the routine automation of de novo 500-mer ODN synthesis. The key to success here is the use of catch-by-polymerization technology to polymerize failure sequences and simplify purification. Graduate and undergraduate students working toward these goals learn a wide range of skills including organic synthesis, nucleic acid chemistry, surface reactivity, automated synthesis, MALDI-MS, electrophoresis and DNA manipulation and sequencing. Inclusive training in these fields and techniques is increasing diversity in STEM fields and in the future workforce.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.