Chemical Synthesis of Long Oligonucleotides

With the support of the Chemical Synthesis (SYN) program in the Division of Chemistry, Professors Shiyue Fang and Yinan Yuan of Michigan Technological University are studying the development of new chemical technologies for the synthesis of long oligonucleotides (ONs), including oligodeoxyribonucleotides (ODNs) and oligoribonucleotides (ORNs). ODNs and ORNs are segments of DNA and RNA molecules, respectively. Long ONs are essential in a wide range of fields, including synthetic biology, protein engineering, gene editing, mRNA therapeutics, gene therapy, nucleic acid vaccines, and computational digital data storage. These fields are closely related to numerous sectors of our society, encompassing energy, environment, agriculture, medicine, and national security. The successful development of the proposed long ON synthesis technologies will enhance the feasibility and cost-effectiveness of projects in these fields. This multidisciplinary research requires a wide range of technical skills such as organic synthesis, surface chemistry, automated DNA and RNA synthesis, NMR, MS, capillary electrophoresis, HPLC, cloning, and DNA and RNA sequencing. During the funding period, Ph.D. students and undergraduate researchers will have the opportunity to acquire these skills through their involvement in the project. The project aims to develop technologies for the de novo synthesis of long ONs. Currently, the longest chemically synthesized ODNs and ORNs are limited to approximately 200-mers and 120-mers, respectively. Longer ONs must be produced using biological methods such as PCR assembly, ligation, and in vitro transcription. These biological methods, however, suffer from drawbacks including long turnaround times, high labor demands, and error susceptibility. More seriously, they are unable to synthesize ONs containing difficult elements such as long repeats, unusually stable higher-order structures, high or low GC content, and site-specific modifications. Recently, the Michigan Tech team achieved direct de novo synthesis of 400-mer ODNs. Building upon this foundation, they will extend the boundaries of chemical ON synthesis to beyond 2,000-mer ODNs and 200-mer ORNs. To achieve these objectives, they will explore novel solid supports for ODN and ORN syntheses and the use of the powerful catching-by-polymerization (CBP) method for the isolation of the low-percentage, yet sufficient quantities, of ONs. The ONs will be rigorously characterized involving techniques such as HPLC, MS, gel electrophoresis, enzymatic digestion, PCR, cloning, and DNA and RNA sequencing. 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.