Team:Rutgers

De novo DNA Synthesis is extremely important to the world of Synthetic Biology, but the low efficiency of today's DNA Synthesis technology limits us.

We can only synthesize about 150 bases at a time, so new genes have to be stitched together from smaller strands, which adds a lot to the time and cost required. This needs to change.

Currently we use organic (anhydrous) solvents and reactive amidites to synthesize all custom DNA sequences, whether for antibody/enzyme engineering, multi-gene pathway building, or even genome construction. The process looks something like this:

This is one cycle in the process of DNA Synthesis. A solution of blocked nucleotides (whichever A, C, G, or T comes next in the sequence) is added to a group of immobilized DNA strands, and a single nucleotide is incorporated at the end of each strand. The trouble with this highly unnatural method is that each of the chemicals depicted (trichloroacetic acid, 2-benzylthiotetrazole, acetonitrile) causes some harm to the chemical structure of DNA, and this will limit the yield in a way that gets exponentially worse when building longer and longer pieces of DNA.

The process pictured above typically causes enough side-reactions to chemically ruin 1.5% of the existing DNA strands in each and every cycle (leaving 98.5% of them intact and with the correct sequence). This concept is called coupling efficiency, and it's the reason we can only synthesize up to 130-150 bases at a time (or even 200 bases if you're willing to pay a whole lot extra).

We believe that both cost and efficiency could be vastly improved by using enzymes for de novo DNA Synthesis. Polymerases have evolved alongside nucleic acids for billions of years and developed beautiful two-metal-ion machinery that makes phosphate diester formation a piece of cake. In theory, this machinery could be applied to de novo DNA Synthesis to eliminate side-reactions and significantly simplify the process overall. A more natural, aqueous synthesis environment would lend itself to higher efficiency, too.

This process simplification could be beneficial on two counts: less complexity in required machinery would reduce capital costs, and less material requirements would reduce operational costs.

This is the enzymatic process that our team has envisioned. The enzyme is a template-independent polymerase called Terminal deoxynucleotidyl Transferase (TdT). It favors single-stranded 3' ends when it adds whatever dNTP's are available. Using the same principles as today's synthesis strategies, it could serve to catalyze the coupling step in each cycle. Upon inspection of this polymerase's binding pocket, it is apparent that even large blocking groups could fit snuggly without hindering the catalytic machinery, but we decided to test a small, simple blocking group for this project to begin with: the acetyl group.

We found that the acetyl group undergoes a bit of background hydrolysis, especially at high pH, so it wouldn't be the ideal blocking group. It does, however, perform well enough to make benchmarking possible so that we can establish a proof of principle for this strategy. We ran many assays, outlined below.

These assays serve to elucidate the functionality of TdT in the conditions of our envisioned DNA Synthesis strategy. We used short, single-stranded primers (each composed of 15 thymidine residues) with 5' fluorescent tags (5'-FAM), and visualized the results on 22% polyacrylamide nondenaturing gels. The images are oriented with the loading wells above, so that the shortest oligos (which travel the fastest) end up furthest down in each lane. Each assay has a Control lane containing only the 15-length primer for reference. All lanes are labeled with neon-green text superimposed over the strange gray blotches of loading dye.