CAS: Long Oligodeoxynucleotides Directly from Automated De Novo Synthesis

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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.