The goal of this project is to advance a novel synthetic oligodeoxynucleotide (i.e. short DNA, typically abbreviated as oligo) purification technology closer to commercialization and widespread utilization in industry. Synthetic oligos and their analogs have wide applications. Here are a few of many examples. They can serve as drugs to cure many diseases including infectious diseases and genetic disorders. The emerging research area ? synthetic biology requires massive numbers of oligos for de novo genome assembly. In the area of digital data storage, scientists predict that oligos can be used as a new medium for data storage and have the potential to solve the problem of data explosion. For these applications, large quantities of a single oligo (e.g. for drug applications) or massive numbers of different oligos (e.g. for genome assembly) are needed. Oligos are typically synthesized on an automated synthesizer. Like any organic synthesis, the product of oligo synthesis contains impurities, which must be removed for almost all applications. Known methods for synthetic oligo purification mainly include HPLC (high performance liquid chromatography) and gel electrophoresis. These methods are unsuitable or not ideal  for large-scale purifications due to drawbacks such as high cost and complicated procedures. Therefore, oligo purification has been a bottleneck in many areas including drug development and synthetic biology.

The new synthetic oligo purification technology to be commercialized utilizes a completely new approach to separate the desired oligo product from impurities. This approach has never been used or tested before our first report in the research area. Using the approach, the purification procedure is drastically simplified, which makes the technology well suited to solve the problems of large-scale oligo purification. The approach is called catching by polymerization, which involves attaching a polymerizable group to the desired oligo product during automated synthesis. The polymerizable group is not attached to the impurities. The attachment is simple and selective. During purification, a simple polymerization reaction is initiated, which activates the polymerizable group on the oligo and co-polymerize the oligo into an insoluble polymer. Because the impurities do not have a polymerizable group, they are not co-polymerized into the polymer. Simply washing the polymer with water removes all the impurities, and highly pure oligo product is obtained by cleaving from the polymer.

With support of this STTR award, the research team has advanced the synthesis of the reagents needed for attaching the polymerizable group to oligos closer to large-scale production, developed detailed protocols for the use of the technology for oligo purification, and proved the high purity of oligos purified with the catching by polymerization technology using a technique called LC-MS (i.e. liquid chromatography coupled with mass spectrometry). In addition, the team also demonstrated the suitability of the new technology for large-scale oligo purification, purification of multiple different oligos simultaneously, and purification of long oligos such as those with more than 300 nucleotides. It is remarkable that the oligos purified with the technology appears as nice crystalline solids (Figure), and are proven to be highly pure with a variety of analytical techniques. The power of the technology for long oligos purification is impressive too. No known oligo purification methods have such a capacity.

Because synthetic oligos are used in many areas, and oligo purification is currently a bottleneck for large-scale oligo production, the new oligo purification technology will have a broad impact in many areas.  Examples include oligo-based drug development, synthetic biology, DNA digital data storage, molecular biology, food industry, agriculture, national security, nanotechnology and more. In addition, the catching by polymerization strategy can also be used for the purification of other biooligomers and their analogs such as RNA, peptide, peptide nucleic acid and oligosaccharides. Biooligomer production is a multiple billion dollar industry. The catching by polymerization purification technology could have the potential to transform this entire industry. This transformation could save substantial societal resources currently needed for biooligomer production. In addition, because the new technology uses much less harmful organic solvents and consumes much less energy, transformation of the industry could have a high impact on our environment. During the funding period, the research team received valuable trainings from the programs organized by NSF on technology commercialization. The experience will help the investigators to commercialize this purification technology and other technologies they are going to encounter in their careers. In addition, through working on the project, two graduate students received training in organic synthesis and nucleic acid chemistry. One of the graduate students is female. Besides typical organic synthesis skills, the students acquired many other skills that are sought by industry. Examples include automated DNA synthesis, phosphoramidite chemistry, polymer chemistry, handling of minute quantities of polar hydroscopic materials, HPLC, MALDI-TOF MS, ESI MS and capillary electrophoresis. Besides graduate students, eight undergraduate students including two female undergraduate students were exposed to synthetic organic chemistry research through this project as well.

Last Modified: 02/20/2019
Modified by: Shiyue Fang