In terms of efficiency, the phosphoramidite method used for synthesizing DNA oligonucleotides is hard to top. Each base-adding step achieves a 99.5% yield. But the method still can’t produce sequences much longer than 300 bases, and because the reaction runs in organic solvents, there’s hazardous waste to contend with. Researchers would like to come up with a method that works in water and makes gene-length sequences of 1,000 bases or more.
A team including Sebastian Palluk, Daniel H. Arlow, and Jay D. Keasling of Lawrence Berkeley National Laboratory reports a new enzyme-based method that they hope will surpass the current gold standard method. The researchers tether a single nucleotide to individual copies of a DNA polymerase called terminal deoxynucleotidyl transferase (TdT) and then use the conjugates to synthesize DNA (Nat. Biotechnol. 2018, DOI: 10.1038/nbt.4173).
The team picked TdT because its job normally is to randomly add single nucleotides to the ends of genes that encode antibodies, a process that helps increase the diversity of the immune proteins made in the body. In the new DNA synthesis method, the enzyme adds its tethered nucleotide to a DNA primer. After adding the nucleotide, the enzyme blocks the addition of more nucleotides until the chemists cleave and wash it away. The researchers repeat the cycle for each nucleotide they want to add.
As a demonstration, the researchers used the conjugates to synthesize 10-nucleotide-long pieces of DNA. The base addition steps had an average yield of ~98%, but the yields varied depending on the base. The researchers will need to optimize the conjugates to achieve the fidelity and yields necessary for synthesizing gene-length DNA, Arlow says.
Michael A. Jensen, a researcher at the Stanford University School of Medicine’s Genome Technology Center, points out that “terminal transferase inherently does not incorporate each nucleotide equally,” which could explain the yield variability. “Further structural modifications to the enzyme itself may be necessary to enhance synthesis efficiency,” he adds.
Despite these potential concerns, Arlow and Palluk have founded a company, Ansa Biotechnologies, to commercialize the technology. The company is in negotiations with Lawrence Berkeley National Laboratory for the intellectual property.