CAS: Long Oligodeoxynucleotides Directly from Automated De Novo Synthesis

CAS：直接来自自动化从头合成的长寡脱氧核苷酸

• 批准号: 1954041
• 负责人: Shiyue Fang
• 金额: $ 49万
• 依托单位: Michigan Technological University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1954041&HistoricalAwards=false
• 关键词: CAS; Long; Oligodeoxynucleotides; Directly; Automated

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.

通过这一奖项，化学系化学合成计划支持密歇根理工大学化学系方世跃博士的研究，开发制造和提纯长寡核苷酸(ODN)的自动化工具。长ODN是用化学方法制备的DNA(2‘-脱氧核糖核酸)分子，含有60个或更多称为核苷酸的单位。它们用于基因组构建、疫苗开发和降低DNA数字存储数据的输入成本。目前，长于200个核苷酸的ODN的制备过程繁琐，需要对许多短的ODN进行低效的制备和纯化，然后采用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造和分离含有500个或更多核苷酸的长ODN，而不会产生大量的溶剂浪费。同样在MTU工作的袁益南博士正在提供ODN表征方面的专家协助。该项目为MTU的不同研究生和本科生以及附近以本科生为主的芬兰迪亚大学的本科生提供跨学科培训机会。制备长ODN的有效方法的发展对合成生物学、核酸疫苗开发和DNA数字存储数据的发展至关重要。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下产生长的ODN。方教授和他的团队正在采取一种整体的方法来实现高效的长ODN合成方法，并通过以下活动满足这一重要需求：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DG丰富区域的偶联产率；3)研究聚合捕捉技术提纯长ODN；4)开发基于MALDI-MS(基质辅助激光解吸电离-质谱仪)的改进的长ODN表征方法；以及5)设计用于长ODN错误的酶校正方案。通过这些目标，方博士和袁博士正在朝着新的500聚体ODN合成的常规自动化方向发展。这里成功的关键是使用聚合捕捉技术来聚合失败序列并简化纯化。为实现这些目标而工作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、表面反应性、自动合成、MALDI-MS、电泳和DNA操纵和测序。这些领域和技术的包容性培训正在STEM领域和未来的劳动力中增加多样性。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。

项目成果

Determination of optical density (OD) of oligodeoxynucleotide from HPLC peak area

从 HPLC 峰面积测定寡脱氧核苷酸的光密度 (OD)

• DOI: 10.7717/peerj-achem.20
• 发表时间: 2022
• 期刊: PeerJ Analytical Chemistry
• 作者: Chillar, Komal;Yin, Yipeng;Eriyagama, Adikari M.;Fang, Shiyue
• 通讯作者: Fang, Shiyue

Oligonucleotide synthesis under mild deprotection conditions

温和脱保护条件下的寡核苷酸合成

• DOI: 10.1039/d2nj03845e
• 发表时间: 2023
• 期刊: New Journal of Chemistry
• 影响因子: 3.3
• 作者: Chillar, Komal;Eriyagama, Adikari M.;Yin, Yipeng;Shahsavari, Shahien;Halami, Bhaskar;Apostle, Alexander;Fang, Shiyue
• 通讯作者: Fang, Shiyue