This Small Business Innovation Research (SBIR) Phase I proposal requests funding to develop a novel technology for the controlled non-templated enzymatic synthesis of single strand DNA molecules for use in molecular, systems, and synthetic biology applications. When implemented, this technology represents the most significant change to the way that oligodeoxynucleotides and polydeoxynucleotides have been produced since the introduction of the phosphoramidite mediated solid support method in 1981. The new process will relieve the bottle necks in molecular and synthetic biology workflows by reducing costs, increasing the lengths of DNA directly synthesized by 10x to 50x, reducing turnaround times from weeks and months to days, and eliminating production of tons of toxic wastes. A unique enzyme will be used that catalyzes the rapid polymerization of deoxynucleotides without need of a template molecule. The project will create novel analogs that are suitable enzymatic substrates and allow the stepwise addition of nucleotides mimicking the gold standard chemical synthesis method but in a simpler and aqueous enzymatic process.
The broader impact/commercial potential of this project, if successful, will be to create a next generation approach to DNA synthesis. Such capabilities will not only enhance current research, but will enable evolving multidisciplinary research areas such as biomedicine, computer science, nano-optoelectronics, and bionanotechnology. Research utilizing DNA nanostructures for drug delivery and single cell in-vivo analysis is evolving. DNA is being used as an information storage medium and for computing itself. Engineers are investigating gene circuits as biological analogs of electronic components such as oscillators and transistors. This most interesting, complex and biologically important molecule surely has many applications that cannot yet be imagined. Just as the first generation DNA synthesis technology enabled a new era of biotechnology 25 years ago, the capabilities presented here will enable a new generation of biological applications and potentially provide for society's future needs.
for NSF Award 1345593 titled "Non-templated enzymatic synthesis of oligodeoxynucleotides" awarded to Molecular Assemblies Inc. In this report we will describe our efforts to fulfill our technical objectives which sought to provide proof of principle for the use of 3'-unblocked reversible terminators for the enzymatically mediated solid support synthesis of long synthetic oligodeoxynucleotides. Our project is innovative and technically risky in so far as successful execution of de novo enzymatic synthesis involves the creation of novel deoxynucleotide triphosphates and their subsequent use in a cyclic synthesis process of user specified single strand fragments of DNA. The introduction of the phosphoramidite mediated method of DNA synthesis in 1981 has revolutionized biotechnology and has enabled, amongst other things, the sequencing of whole genomes and the introduction of powerful PCR based analysis, both of which are allowing unprecedented insight into the biology of human, plant and animal systems. This era of "reading" genomes is now leading to the rise of a new era based on the "writing" of genomes as basic research and industry desire to alter the output of genes, entire metabolic pathways and whole genomes for useful outcomes. With an average length of a human gene being 1500 bases in length, this use of synthetic DNA has placed new demands on the cost, length and quality constraints of the existing chemical process. While significant effort and hundreds of millions of dollars has gone into parallel synthesis using arrays, miniaturized DNA synthesizers and methods to "fish out" the desired products from complex mixtures, there has been little effort to change the paradigm of the basic approach to synthesizing DNA. Molecular Assemblies was founded to develop a next generation DNA synthesis process based on the use of enzymes for an aqueous based DNA synthesis method which if successful will allow the synthesis of longer molecules at a lower cost and without the use of hazardous organic solvents and reagents. The proof of principle of our approach requires the synthesis of novel 3'-unblocked reversible terminators and the demonstration of their use in repetitive cycles of DNA synthesis on a solid support. During the 6 month period of the award, we were able to establish some of the key intermediate steps required for the modification of nucleosides and their conversion into triphosphates. We were also able to demonstrate that several nucleoside analogs of the type that we are interested in, retain substrate activity with the commercially available template-independent enzyme that we have chosen as the engine of our process. We have also begun development of a solid support model system which will be the primary assessment tool for the achievement of our technical goals and have shown that enzymatic synthesis of DNA is possible on magnetic beads. We expect to complete proof of principle by the end of 2014. While not directly attributable to our efforts during this Phase I SBIR, Molecular Assemblies was granted a patent, US 8,808,989 further validating the novelty of our approach. While there is still more work to be done to realize the goal of enzymatically mediated de novo syntheis of DNA as a potential replacement for the use of chemically synthesized DNA in applications like metabolic engineering, synthetic biology, systems biology, and DNA-based nanotechnology, Molecular Assemblies has made significant progress based on the award received from the NSF. The technical progress that has been made during this six month period is enabling the infusion of additional funding that will allow the completion of the development work required for the ultimate commercialization of the next generation of DNA synthesis.
