SBIR Phase I: Reagent Development for a Rapid Enzymatic DNA Synthesis Platform

The broader impact/commercial potential of this Small Business Innovation Research (SBIR) project is to advance the development of a new method for rapid and accurate DNA synthesis. The ability to synthesize "gene-length" DNA molecules (>500 bases) is invaluable for biological and biomedical research and is a key enabling technology for the rapidly growing field of synthetic biology. Existing methods for producing DNA sequences rely on organic chemistry and are generally limited to the direct synthesis of 200 base molecules, necessitating further assembly into gene-length products. However, the assembly process used is failure-prone and not compatible with all desired sequences. The technology being developed in this project will demonstrate the development of a new DNA synthesis method based on enzymes, which, in contrast to organic chemistry, promises to enable the direct synthesis of gene-length DNA molecules. An additional benefit of enzymatic DNA synthesis is that it is performed in aqueous conditions using nontoxic reagents, whereas chemical DNA synthesis uses toxic reagents and produces hazardous waste. The enzymatic DNA synthesis method, once fully developed, will be the core technology supporting a rapid DNA synthesis service that will accelerate research in life sciences and synthetic biology. This SBIR Phase I project proposes to develop optimized "TdT-dNTP conjugate" reagents for a new enzymatic DNA synthesis method. The exquisite specificity of enzymes promises to enable the direct synthesis of much longer DNA constructs than is possible with the industry-standard chemical DNA synthesis method in use today, accelerating workflows in synthetic biology. A TdT-dNTP conjugate consists of the "template-independent polymerase" enzyme TdT with a tethered nucleoside triphosphate substrate that can be incorporated into a DNA molecule by TdT. After adding the tethered nucleotide to a DNA molecule, the polymerase remains attached to the end of the DNA molecule via the tether to the added nucleotide, blocking other conjugate molecules from further extending the DNA molecule, and thus ensuring that the molecule only gets extended by one nucleotide. The linkage can then be cleaved to release the polymerase and expose the DNA molecule for subsequent extension. Previous research has demonstrated that TdT-dNTP conjugates based on a photocleavable linker can be used for stepwise de novo DNA synthesis. This project proposes to develop new TdT-dNTP conjugates based on a linker that can be rapidly and specifically cleaved using a hydrolase enzyme, finally realizing the promise of a fully-enzymatic DNA synthesis method. 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.