“The state of DNA synthesis is a bit like humanity’s journey to the moon—just because something has been achieved before doesn’t mean we should cease figuring out how to do it better.”

That is how Harold P. de Vladar, PhD, founder and CEO of synthetic DNA provider Ribbon Biolabs, sees the vast opportunity in the DNA synthesis space. de Vladar says that the DNA synthesis equivalent to the Apollo 11 moon landing was J. Craig Venter’s landmark work on assembling the 5,386-bp bacteriophage FX174 from short single strands of synthetic, commercially available DNA known as oligonucleotides. Venter and his team at the J. Craig Venter Institute built the FX174 genome using a polymerase chain reaction (PCR) adaptation called polymerase cycle assembly. The feat occurred in 2002–2003—just after the sequencing of the human genome in the early 2000s.

Venter showed that long DNA could be synthesized, but he also confirmed that doing so was difficult. “When I was still a researcher,” de Vladar recalled, “I confronted the problem of making long DNA. I wanted to synthesize a library of small phage genomes, around 4,000 bp, and that was an impossible project. I wanted like 250 sequences, and this was unachievable.”

In 2018, de Vladar launched Ribbon Biolabs in Vienna, Austria. Ribbon needs just one day to reliably construct 1-kb fragments that can be assembled into larger molecules of 10–20 kb. To do so, Ribbon algorithmically processes a sequence into smaller pieces that get sorted into a decision tree. This allows Ribbon to identify the best combination of 10- and 12-mer oligonucleotides (from the company’s biobank of nearly 80,000 oligonucleotides) for automated assembly. The true turnaround time, including sequencing-based verification and delivery, is about three days. The goal for de Vladar and Ribbon is to scale the gene synthesis from the library approach to 1,000 kb per day.

Assembly versus synthesis

In the past decade, many powerful DNA assembly methods—including Gibson assembly—have been developed. These methods have been applied in the construction of a minimal bacterial genome and synthetic yeast chromosomes.

Assembly approaches to DNA synthesis have several drawbacks: the potential for contamination, the dependence on sequence confirmation from an individual clone, and the reliance on high-fidelity proofreading PCR enzymes, which must be used to copy constructed genes to prevent mutations during amplification.

But not everyone agrees that assembly is DNA synthesis. “When we talk about DNA synthesis, we have to be a little cautious,” says Sylvain Gariel, co-founder and COO of DNA Script, a company that chemically synthesizes DNA. “Some people talk about oligonucleotide synthesis, and other people talk about gene assembly.”

Over the decades, scientists have developed various methods for generating synthetic DNA. For example, chemical methods have been developed that can reliably provide short DNA chains, typically <200 nucleotides (oligonucleotides). These methods work best with automatic synthesizers, which are now needed for gene engineering and sequencing.

Scientists have also developed enzymatic methods, which can generate DNA that is longer and more complex than the oligonucleotides generated with chemical methods. Enzymatic methods can work with or without DNA templates. For instance, the basic technology for DNA Script is template-independent enzymatic oligonucleotide synthesis.

Companies have turned these synthesis methods into products and services, making synthetic DNA available to people who are not experts.

On March 9, 2023, Ansa Biotechnologies reported that it had successfully made the world’s longest DNA oligonucleotide in a single synthesis. The 1,005-base sequence codes for a key part of an adeno-associated virus vector used to develop gene therapy. Because the sequence has complex features, such as strong secondary structures and high GC content, it is very hard to make with traditional methods, which require that shorter oligonucleotides be put

Evonetix Chip Plus People
Evonetix has developed semiconductor technology that allows thousands of independent DNA sequences to be synthesized at thermally controlled reaction sites across the surface of a single chip. The technology also allows long DNA molecules to be assembled while synthesis errors are removed. The wafer shown here holds multiple 24 × 21 mm chips, each of which becomes part of a synthesis cartridge along with the reagents necessary for a single synthesis run within a benchtop instrument. The instrument manages the electronic and fluidic connections needed to prepare the DNA sequences specified by the user. All synthesis operations are fully automated.

Do-it-yourself DNA printing

As a metabolite engineer, Gariel needed to acquire DNA constructs, which were seldom as readily available as he would have liked. He had to wait weeks or even months to obtain long stretches of DNA for tens or even hundreds of genes. Eventually, he became interested in bypassing this bottleneck through the development of a benchtop DNA synthesis device.

Gariel summarizes the rationale behind DNA Script’s early work as follows: “I’m suddenly cutting out all the time that the service provider requires. I’m not giving control to a service provider as long as I have an instrument and the reagents that I need to run it. I can do it on the benchtop without any specific infrastructure requirements other than a molecular biology space and an electric plug.”

Ultimately, DNA Script developed a benchtop DNA synthesis device called Syntax. To make DNA synthesis safer and more accessible, DNA Script has addressed the danger posed by phosphoramidite chemistry, both to the user and the environment. Gariel notes that this chemistry comes with strong constraints in terms of infrastructure and dedicated labor.

Instrument Plus Workflow
The Syntax system from DNA Script is a benchtop enzymatic DNA synthesis (EDS) platform. It employs a cyclic two-step process to enable the on-demand synthesis of custom DNA oligonucleotides without the need for specialized infrastructure or technical skills. Step 1 is for elongation. Terminal deoxynucleotidyl transferase adds a single nucleotide to a predefined initiator DNA that is attached to a solid support. (This allows for the control of synthesis scale and the manipulation of the growing oligonucleotide strands throughout the EDS process.) Step 2 is for deprotection. The reversible terminator of the nucleotide is removed, leaving the strand ready to be elongated again. Steps 1 and 2 are repeated until the longest oligonucleotide on the plate is completed.

Matthew Hayes, PhD, co-founder and CTO of Evonetix, says that most attempts to make DNA have focused on shrinking the technology. Evonetix, he points out, has taken a very different approach, one that raises fundamental questions: What’s wrong with DNA synthesis? Why is it fundamentally difficult to make long pieces of DNA? Can we come up with a solution that solves that from the ground up?

“There are people today who can make machines that will make a small number of high-yield oligonucleotides and PCR primers using column-based synthesis,” Hayes says. “But if you want an array of low molecular quantity and a high diversity pool, you’re pretty much restricted to service providers.” Evonetix plans to give users instruments that use semiconductor-based chips to synthesize oligonucleotide pools. “We’re able to instruct our machine to assemble an oligonucleotide pool into a much longer double-stranded DNA template,” Hayes asserts.

Chemical DNA synthesis as a service

This is not the first time that benchtop DNA synthesizers have hit the market. Emily Leproust, PhD, CEO of Twist Bioscience, recalls when people stopped buying benchtop DNA synthesizers. She started her doctoral work in DNA synthesis in 1996, and she bought one of the last decentralized desktop oligonucleotide synthesizers in 1998. “A lot of people have forgotten that we used to have the benchtop DNA synthesizer,” she says. “People stopped buying DNA synthesizers [because] it was just cheaper and faster to send out the work of DNA synthesis.”

Leproust asserts that Twist, a DNA synthesis company, offers unlimited scalability. “When I had my desktop DNA synthesizer in graduate school, I could make eight oligos,” she relates. “Now, desktops can do around 96. But what if you want to make 500 oligos? You have to run the instrument five times in a row. If you want 10,000 oligos, you have to run it 100 times in a row! Our range is super flexible.”

DNA synthesis companies offer the ability to make large amounts of complex DNA quickly at prices that are finally becoming more affordable. Twist’s silicon- based high-throughput gene synthesis platform makes high-quality gene fragments for as little as $0.07/bp and perfect clonal gene sequences verified by next-generation sequencing for as little as $0.09/bp.

“Our technology can make millions of oligos at the same time, up to 300 bases with a quality of about one error in 2,000–3,000 bases, and we can assemble up to 5,000 bases in a construct,” she says. “If you want fragments of 1 kb in length, we can do that for $70 and ship them to you in four days, and we can make more than 1 million of these 1,000-base fragments a year.”

Although Leproust thinks that Twist’s technology can push past 300-base oligonucleotides, the synthesis of the oligonucleotides is relatively slow compared with stitching them together. “If we were to make 1,000 bases all by synthesis, it would not be faster than what we shipped today in four days,” Leproust points out. “And you’ll lose out on speed, quality, and cost.

“There’s some vanity in saying, ‘I can make a 1,000-bp fragment.’ But if it’s more expensive, slower, and of lower quality, that’s not what the customer wants. We’re more into what moves the needle for the customer. I’m not into running science experiments.”

Twist Chip Plus People
Twist Bioscience has developed a proprietary semiconductor-based synthetic DNA manufacturing process that features a high-throughput silicon platform. According to Twist, the platform reduces reaction volumes by a factor of 1,000,000 while increasing throughput by a factor of 1,000, enabling the synthesis of 9,600 genes on a single silicon chip at full scale. Traditional synthesis methods produce a single gene in the same physical space using a 96-well plate.

Hot take: Protein writing

When a technological field is becoming increasingly crowded, the most obvious way to take the lead is to make things orders of magnitude better than the state of the art. But in the DNA synthesis field, that option didn’t seem available. “We didn’t see a trajectory where enzymatic DNA synthesis would result in a 10-fold  or 100-fold improvement in something for the customer,” says Michael Chen, PhD, a co-founder and the CEO of Nuclera. So, Chen and his fellow co-founders decided that Nuclera should try something else. Nuclera began focusing on addressing a key bottleneck in enzymatic DNA synthesis: the making of proteins to write DNA.

Chen saw an opportunity to take Nuclera’s core digital microfluidics technology and collaborate with E Ink, a developer of electrophoretic display products. In fact, Nuclera acquired E Ink’s digital microfluidics unit.

“The supply chain and research to make electrophoretic displays have a lot to do with the type of droplet automation technology that we’re working on and commercializing at Nuclera,” Chen explains. “We’ve made [the eDrop lab-on-a-chip technology] to move thousands of droplets in a programmable fashion in what we call a ‘pipette and forget’ process. The researcher pipettes directly into the cartridge and then can walk away.”

A virtuous cycle

In the synthetic DNA field, there are two mutually reinforcing developments. First, there is rising demand for the mass production of long synthetic DNA. Second, there is a surge in the development and commercialization of DNA synthesis technologies. Each development intensifies the other.

Rising demand is evident in various areas. The most established areas include biologics and synthetic biology. Emerging areas include vaccines, gene therapies, DNA data storage, DNA origami, nanobots, and plants that are resistant to climate change. Progress in these areas is driving (and is being driven by) improvements in DNA synthesis technologies.

Not all of the DNA synthesis solutions that have been developed have reached the market, but service-oriented and do-it-yourself approaches already seem viable. Moreover, both approaches are improving in terms of price, speed, and quality.

This article was originally published in the April 2023 issue of the GEN Biotechnology journal. GEN Biotechnology, published by Mary Ann Liebert, Inc., is a marquee peer-reviewed journal publishing outstanding original research and perspectives across all facets of the biotech industry.

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