DNA printing gets an upgrade

The process uses no toxic chemicals and promises to produces DNA strands 10 times longer than today’s methods.

Some of the most exciting things happening in synthetic biology, from engineering organisms to breakdown plastic to making biofuels and medicines, all require custom DNA sequences. Producing these sequences is currently slow and expensive, creating a critical bottleneck to research progress. Now, researchers have found a new way to synthesize DNA that works by harnessing an enzyme called TdT (terminal deoxynucleotidyl transferase).

Other labs have also tried to use TdT in the past, because it is one of few enzymes that writes new DNA from scratch rather than copying it. The problem with the enzyme is that it’s very hard to control. To make the desired custom DNA sequence, the enzyme needs to add just one nucleotide, or DNA building block, and then stop before it keeps adding the same nucleotide repeatedly. To stop it from repeatedly adding the same nucleotide, some labs have tried to add a blocking nucleotide, but this can interfere with the activity of the enzyme. So researchers Dan Arlow and Sebastian Palluk decided to instead tether one nucleotide to TdT and cut the linking tether after the DNA molecule has been extended.

We spoke with one of the authors of the paper, Dan Arlow, about the work.

ResearchGate: Can you tell us how this study came about?

Dan Arlow: Sebastian and I were working on separate synthetic biology projects before we met—me in the Keasling Lab, and Sebastian at TU Darmstadt. We were both frustrated by how long it took to get synthetic genes manufactured for our research projects. Independently, we both came to the conclusion that an enzymatic DNA synthesis method was needed to overcome the limitations of the chemical method currently used to synthesize DNA. Sebastian's friend from home was doing a master’s thesis in the Keasling Lab, and when he heard that I was also passionate about new methods for DNA synthesis, he introduced me to Sebastian. We hit it off, and Sebastian came to Joint BioEnergy Institute (JBEI) to work with me in 2015. We got the key idea behind this project in late 2015, and Sebastian quickly collected the first key proof-of-concept result. We have since worked together on every aspect of this project.

RG: How is DNA typically synthesized, and why does it need to be improved?

Arlow: Synthetic DNA is currently produced using the “nucleoside phosphoramidite” method, which was first developed in the early 1980s by Marvin Caruthers and colleagues. The development of that method marked a key point in the biotechnological revolution and has withstood the test of time. In the method, growing DNA molecules immobilized on a solid support (typically glass) are extended using activated monomers with removable blocking groups to synthesize a desired sequence one base at a time. The reactions mostly take place in organic solvents, and when performed under carefully optimized conditions, have stepwise yields exceeding 99.5 percent.

However, in practice it is very difficult to synthesize DNA molecules longer than 150-200 nucleotides using the method, in part due to side reactions. As such, longer DNA molecules must be divided into overlapping short sequences that are synthesized separately and then stitched together into the full-length product. Unfortunately, the stitching process can introduce errors and is not amenable to all desired sequences.

RG: And how does your enzyme-focused approach improve on this?

Arlow: There has been longstanding interest in enzymatic DNA synthesis methods because enzymes can be exquisitely precise and essentially eliminate side reactions. This might enable the direct synthesis of longer sequences than is currently possible with the phosphoramidite method. Also, enzymes operate in aqueous conditions and need not produce hazardous solvent waste.

RG: Is your work and other efforts to synthesize DNA, like Ginkgo Bioworks, evidence of a larger push to improve this process? Why is it so important?

Arlow: We sense that there is growing understanding that DNA synthesis is a bottleneck in biological research and engineering. I think that synthetic biology labs and companies probably feel the need for improved DNA synthesis methods most acutely, but faster DNA synthesis will likely accelerate research and spur innovation throughout life sciences, including in therapeutics, diagnostics, and industrial biotechnology. Personally, I am most excited about applications in sustainable biomanufacturing of useful products and materials, because it promises to reduce our impact on the environment.

Featured image courtesy of Mehmet Pinarci.