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Why are we TdT-free?

Why are we TdT-free?
3 minute read


In recent years, there has been significant interest in using an enzyme called Terminal deoxynucleotidyl transferase (TdT) to produce synthetic DNA. If we're a cutting-edge DNA synthesis company, why aren't we using TdT?

Phosphoramidite chemistry has been the gold-standard DNA synthesis technology since the 1980s. But now, researchers' demand for synthetic DNA highlights the limitations of this approach. The image at the top of this post clearly shows the visible by-products of phosphoramidite synthesis.  

TdT DNA synthesis: real or rhetoric?

Getting your hands on custom, short, synthetic DNA sequences is largely straightforward. You place an order online, and as long as your DNA sequence is less than 200 or so nucleotides and it meets a defined complexity score, then it can be chemically synthesised. It's also likely to be on your doorstep in a matter of days. 

But what happens when you require longer, more complex DNA? Newer applications and therapeutics, like CRISPR, mRNA vaccines, and cell and gene therapies, set their sights on lengthy and complex sequences that are beyond the reach of the phosphoramidite process. 

Multiple start-ups are exploring the potential of enzymatic synthesis as a faster and more efficient strategy, addressing the limitations of phosphoramidite synthesis. Most have honed in on a polymerase enzyme called terminal deoxynucleotidyl transferase (TdT), causing quite a stir in the scientific ether. 

Our first attempt to improve DNA synthesis used a TdT method, but we abandoned that approach. 

Controlling TdT's function

TdT’s biological function is to generate antigen receptor diversity and in turn, expand the diversity of foreign bodies our immune system can detect. It does this through its function as a DNA polymerase.

There are a wide number of studied DNA polymerases, with many functioning to rapidly copy a strand (template) of DNA. And they're fast enzymes, copying as many as 1,000 nucleotides per second. Enzymes like TdT are template-independent DNA polymerases, so if they are controlled, they could produce custom synthetic DNA sequences.

DNA polymerases have been developed for a wide range of applications within biotechnology. A good example is sequencing-by-synthesis (SBS), which is the basis of next-generation DNA sequencing. The approach works by controlling the addition of just one nucleotide at a time that, in the case of SBS, is also labeled with a fluorescent dye. 

TdT polymerises DNA by adding new nucleotides in a 5' - 3' direction. In theory, this can be controlled by only using modified, non-natural, nucleotides, which have a reversible block at their 3' end. However, we discovered significant challenges with this approach.

In its normal biological function, TdT has a bias toward the incorporation of G and C nucleotides, which is thought to contribute towards the stability and affinity of antigen receptors for their targets. It also has a stronger preference for natural nucleotides compared to the modified ones.

An unrealistic technology challenge

TdT's function may be important for creating antigen diversity, but it creates challenges for synthetic DNA production, where we want complete control over which nucleotide is incorporated. The preference for incorporating some nucleotides over others compromises the reliability of DNA synthesis and the efficiency with which certain sequences can be produced. 

As a result:

  • In our hands, the TdT approach was more error-prone than phosphoramidite synthesis.
  • A more error-prone de novo synthesis method reduces the ability to produce complex gene-length DNA sequences.  

Enzyme synthesis that catalyses your DNA production

Synthetic DNA is used every hour of every day in research labs and drug manufacturing. Despite this, building DNA constructs, gene-length DNA, or complex sequences is slow and frustrating work. We realised that TdT-synthesis would not compete with phosphoramidite synthesis, but it did lead us to develop gSynth, a TdT-free DNA synthesis technology. 

gSynth harnesses the power of enzymes to accurately produce DNA sequences of any length and complexity. Its unique enzyme mix, novel building blocks, and pioneering process have given rise to a technology that is: 

  • Unbiased: Our uniquely optimised mix of enzymes and proprietary bioinformatics algorithm, Cimban™, incorporate the nucleotides you want, where you want them. 
  • Flexible: gSynth’s technology produces dsDNA and effortlessly handles any secondary structures that come its way!

Looking for a more accurate, more sustainable way to scale long and complex DNA production?

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