The main goal of this project was to develop a chemical method capable of synthesizing oligos (i.e. segments of DNAs) with unprecedented lengths. There are many reasons for the need for chemical synthesis of long oligos. One of them is that, unlike biological methods, it does not require a DNA template. This capability is particularly important for projects in fields such as synthetic biology, protein engineering, and the development of mRNA vaccines and therapeutics, where oligo sequences often need to be designed and synthesized de novo due to the lack of existing templates. In addition, using chemical method, oligos with site-specific natural or unnatural modifications can be synthesized, which is not possible or difficult for biological methods. The state-of-the-art chemical methods can only synthesize oligos with about 200 units (i.e. nucleotides) or shorter, and the quality is usually low (e.g. high error rate, low yield) when the length approaches 200 units. However, many applications including those mentioned above need longer oligos, and for some applications such as synthetic biology, the longer the better.

We planned to synthesize oligos with 500 units or longer. After four years of intensive experimentation, marked by many setbacks, we achieved our goal. We successfully synthesized genes with up to 1,728 units, far exceeding the original goal of 500 units. The sequences of the long oligos were confirmed using reliable analytical methods, including gel electrophoresis, cloning, and Sanger sequencing. The innovations that enabled the breakthrough include conducting oligo synthesis on smooth surface instead of within pores of solid supports, as is typical of known synthesis methods, and employing the powerful catching-by-polymerization method to isolate the low percentages of desired sequences from complex mixture, for example, 100 picomole oligo from a 1 micromole synthesis, or 0.01%. It is noted that for many biological applications, 1 picomole oligo is sufficient.

We expect that the long oligo synthesis method will have a notable impact on projects across many areas, including those mentioned above. One of the major challenges in these fields is obtaining long oligos or DNAs with high sequence accuracy. For this reason, substantial resources have been devoted to the development of enzymatic methods that do not require a template for long oligo synthesis, and de novo synthesis of 1,000 units oligos has long been a dream. Now that dream has been realized, although using a chemical method, it is hopeful that the lives of researchers who need long oligos will become easier.

In addition to achieving the main goal of the project, the research team made several other significant contributions. These include studying the effects of epitranscriptomic RNA modifications on SARS-CoV-2 life cycle, developing a MALDI MS method capable of analyzing long oligos, and developing a method for synthesizing sensitive epitranscriptomic RNAs that cannot be synthesized, or are extremely difficult to synthesize, using any known methods.

In terms of broader impacts, long oligos are essential for projects in areas such as synthetic biology, protein engineering, mRNA therapeutics, and many others. These areas influence numerous aspects of society, including the economy, energy, environment, agriculture, health, medicine, national security and more. In the funding period, the research team, comprising the groups led by Dr. Shiyue Fang and Dr. Yinan Yuan, trained seven PhD students, four MS students, and 16 undergraduate researchers. Among them, three have graduated with PhD, and four have graduated with MS degrees. The group included five black students, eleven female students, and two students from Finlandia University, a nearby primarily undergraduate institution. The students gained a wide range of skills, including but are not limited to organic synthesis, nucleic acid chemistry, automated DNA/RNA synthesis, HPLC, NMR, ESI and MALDI MS, capillary electrophoresis, PCR, gel electrophoresis, cell culturing, cloning, DNA isolation, and DNA sequencing. These individuals are valuable assets to society. For instance, the three PhD graduates are now playing crucial roles in biotech companies, contributing to innovations in biotechnology and next-generation medicine at the interface of chemistry and biology. 

Last Modified: 12/12/2024
Modified by: Shiyue Fang