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Off to a catalytic start: Enzymatic DNA synthesis

We all probably remember the enzyme kinetics graphs from school about optimal pH, temperature and substrate concentration in relation to rate of reaction. Well, enzymes are vital biological molecules (typically proteins) that significantly speed up chemical reactions within cells. They play key roles in a number of processes within the body, including digestion and metabolism. On the other hand, these molecules have also provided use in vitro.

The most commonly used form of enzymatic DNA synthesis is polymerase chain reaction (PCR). Experts widely use PCR to rapidly make copies of specific DNA samples, which is particularly important when there is very little sample available. For decades, biologists have been building custom DNA sequences chemically. However, new enzymatic strategies are emerging that help circumvent some of the limitations. In this blog, we explore what enzymatic DNA synthesis is and what promise it holds for the future.

What is enzymatic DNA synthesis?

Standard approach

In recent years, the field of synthetic biology has garnered a lot of interest. This field of science involves the application of engineering principles to biology. It seeks to create new biological parts, devices and systems or to redesign systems that already exist in nature. However, synthetic biology relies on advances in DNA synthesis for easy, affordable and quick access to accurate nucleic sequences.

While there have been several advances in sequencing methods and approaches, our ability to write DNA has fallen short. Standard DNA synthesis continues to rely on a 40-year-old phosphoramidite chemistry. This process involves multiple rounds of stepwise assembly of chemically modified nucleotides. However, this chemistry is limited in its ability to produce long DNA of sufficient quality, with efficiency dropping off beyond ~200 mers. Not only this, the harsh chemicals used are also environmentally hazardous.

Enzymatic approach

Luckily, template-independent DNA synthesis is well established in nature. Most researchers have homed in on a specific DNA polymerase that requires no template – terminal deoxynucleotidyl transferase (TdT). Researchers realised the theoretical potential of this enzyme as tool for controlled DNA synthesis in the early 1960s. However, the exact mechanism and function of the enzyme remained poorly understood for several decades. Then around 2013 to 2014, the need for DNA synthesis alternatives fuelled a surge of interest. Subsequently, three companies offering TdT-based DNA synthesis were founded – Molecular Assemblies, DNA Script and Nuclera.   

Controlled synthesis of specific DNA sequences occurs somewhat like sequencing by synthesis (SBS) – used in several NGS platforms. The same TdTs can be used as catalysts in combination with four reversible terminator deoxyribonucleotide triphosphate (dNTP) analogues. However, unlike SBS, this method does not require a template nucleic acid and is capable of de novo synthesis. After multiple cycles of extension, the strand can then be cleaved from its solid support. Since the DNA is synthesised through an enzymatic process, it exists as a completely natural, biologically active molecule.

The future of enzymatic DNA synthesis?


Although promising, several challenges in enzymatic DNA synthesis remain. Researchers have noted that the enzyme exhibits some notable biases. This in turn could affect the reliability of the process. The performance can also decline if the strand starts to form secondary structures. Therefore, more protein engineering may be necessary to improve the enzyme’s performance and increase its reliability.


There are at least half a dozen companies now active in this space. As a result, a variety of business models have emerged. Nuclera and DNA Script are both working on developing benchtop DNA printer instruments. Additionally, a few companies have already started manufacturing enzymatically generated DNA for customers (yet on a limited basis). For example, Camena is producing oligos for a COVID-19 test currently in development.

Beyond synthetic biology, this method could also help accelerate the production of nucleic acid-based vaccines. Excitingly, researchers could also use it for DNA-based data storage. For example, Twist Bioscience last month stored a Netflix Original Series on their synthetic DNA. However, the goal here is somewhat different. Using DNA to store information will also require advances in synthesis throughput and reductions in the cost per base.


After years of effort, enzymatic synthesis performance is reaching some important milestones. For example, DNA Script has managed to achieve up to 99.7% coupling efficiency with its enzymatic process. This exceeds the estimated 99.2-99.3% efficiency of phosphoramidite chemistry.

Enzymatic synthesis of DNA is still in its infancy, but there has already been a lot of progress and innovation that has emerged from the field. There are several areas of interest emerging from this technique including large-scale synthesis of nucleic acids for therapeutic, agricultural and nucleic acid-based materials and also DNA-data storage.

If the core fundamentals of enzymatic synthesis translate effectively in these applications, the ability to develop fast, high-throughput synthesis of long DNA strands on demand could be revolutionary.

 Image credit: By Yuuji –

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