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Twist Bioscience Sees Demand for New Enzymatic DNA Synthesis Method; Stays Mum on Timeline, Details

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BALTIMORE – With a novel enzymatic DNA synthesis method announced last week, Twist Bioscience is poised to step into an increasingly crowded market, but the firm is providing no timeline or technical details at this time.

The announcement, made by CEO Emily Leproust at the 2022 JP Morgan Healthcare Conference, comes after several other firms revealed their plans for developing enzymatic DNA synthesis in recent years. Twist said it has spent the past 18 months developing a new way to manufacture DNA enzymatically, claiming the method to be "innovative, low cost, scarless, and scalable." The approach will eventually find applications in DNA storage, PCR primer and probe production, plasmid DNA synthesis, qPCR assays, and decentralized DNA synthesis, according to the firm.

Compared with traditional phosphoramidite DNA synthesis, which relies on harsh reagents and can only produce oligos about 200 to 300 nucleotides in length, enzymatic DNA synthesis has the potential to make longer DNA strands using less toxic chemicals.

Named Twist Enzymatic DNA Synthesis 3.0, Twist’s new technique uses a linker that connects a polymerase with a nucleotide and is purportedly cheaper than other methods because it eliminates the need for excess deoxynucleotide triphosphates, or dNTPs, during synthesis. According to Leproust, Twist scientists also discovered a linker that can "circumvent the issue of scarring, so that the DNA synthesized using our process is identical to natural DNA."

But according to John Nelson, a senior principal scientist for GE who also works on enzymatic DNA synthesis, linking enzymes with nucleotides does not necessarily lower the production cost. This is because even if attaching enzymes to nucleotides can eliminate the need for excess free-floating dNTPs, a large concentration of enzyme-nucleotide complex is still likely needed — and subsequently washed away — to achieve high efficiency during the reaction. "The more [nucleotides] you put in, the greater the probability that 100 percent of your oligos are going to get extended," he said.

To make it less wasteful, Nelson added, Twist needs to have "a very efficient enzyme-linked-to-nucleotide system, so they don't have to flood the system with enzymes in order to get the reaction to happen quickly."

Twist said it does not plan to disclose any specifics about the enzyme or linker before its patent application becomes public, but Leproust noted that the method still needs "a lot of enzyme engineering" to achieve comparable oligo length and quality to its chemical synthesis. She also did not share a timeline for releasing the technology.

In the meantime, several other companies, including French startup DNA Script; University of California, Berkeley-spinout Ansa Biotechnologies; and San Diego-based Molecular Assemblies, have been developing methods for making oligos with enzymes in vitro.

DNA Script, for example, which recently raised $200 million in Series C financing, plans to launch its CE-marked Syntax enzymatic DNA synthesis platform in the US and Europe early this year.

Current enzymatic oligo synthesis methods use terminal deoxynucleotidyl transferase, or TdT, a vertebrate polymerase known to scientists for decades that can add single nucleotides to the 3'-end of single-stranded DNA without a template.

In the case of DNA Script, the workflow starts with a highly engineered TdT binding to an initiator DNA that is anchored on a solid support in a reaction chamber. While the system is flooded with dNTPs, the enzyme incorporates a single nucleotide that has a reversible terminator at the 3' end into the DNA strand. In the next step, the blocking group is removed from the DNA, the reagents are washed away, and the cycle repeats.

Because TdT tends not to work efficiently with 3'-blocked nucleotides, other companies, such as Ansa, tether a single nucleotide to a TdT enzyme through a molecular linker. After the enzyme adds the nucleotide to a DNA molecule, it remains covalently attached to the DNA, thus preventing any further additions. After cleaving the enzyme off, the next nucleotide can be added. Unlike Twist's approach, the process leaves a tiny molecular "scar" on each base of the newly synthesized DNA, however, making it not completely natural.

Leproust said that following the launch of its new method, Twist intends to keep both ways of DNA production, and both can be implemented on its proprietary silicon-based platform. "Twist's platform is chemistry agnostic, so we can switch to enzyme quickly," she said. "We're not married to [chemical synthesis]."

In addition to offering enzymatic synthesis as a service, the company is open to licensing the technology out. "We think that enzyme[-based synthesis] opens up an opportunity for decentralized DNA synthesis," Leproust said. "We are very happy to partner with others."

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