Friday, April 26, 2024
4/26/2024
ABSTRACT The objective of this proposal is to develop a benchtop, inkjet-based, oligonucleotide synthesizer using aqueous, enzymatic DNA synthesis technologies for the parallel synthesis of up to 1 million high-quality oligonucleotides per day. The system will deliver >10-fold improvements in the key metrics of cost, quality, and throughput compared to existing technologies. The synthesis biochemistry will utilize engineered terminal deoxynucleotidyl transferase enzymes that have been optimized for incorporation of 3’ aminoxy reversible terminator nucleotides. This approach has several advantages, including proven performance in the synthesis of oligonucleotides up to 300 nucleotides on microbeads. Here, we will adapt the technology to enable DNA synthesis on functionalized microscope slides using inkjets for precise reagent delivery. A multidisciplinary approach will be followed to advance toward the goals in terms of engineering for instrumentation development, biochemistry and protocol optimization, and performance benchmarking of the synthesized oligonucleotides. During the first year, we will build an initial testbed to enable verification and optimization of individual components, materials, reagents, and protocols. In the second year, we will build a fully automated prototype instrument, and refine the protocols and software to allow synthesis of long oligonucleotides. Finally, Year-3, we will build multiple benchtop instruments and work with partner laboratories to test the oligonucleotides in several ‘real world’ applications. Subsequent commercialization of the instruments will democratize access to rapid high-density oligonucleotide synthesis, and revolutionize approaches to a wide variety of synthetic biology and functional genomics applications.
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