TdT - A story about persistence

This article was originally published here.

Last week, I talked about the latest news from the enzymatic DNA synthesis space. This week, let's take a dive into the origin of the most promising enzyme candidates to write DNA from scratch (de novo) - terminal deoxynucleotidyl transferase (TdT, yes it's a bit of a mouthful).

The conception of using TdT to make DNA de novo actually predates our ability to make DNA using phosphoramidite-based chemistry. In 1959, when American scientist Frederick James Bollum was, in his own words, "fumbling around with eukaryotic DNA polymerases" isolated from the calf thymus gland, he discovered an enzyme that does not require a template to elongate single-stranded DNA. Like all great scientific discoveries, his colleagues at Oak Ridge National Laboratory were not convinced, and "preferred to interpret the result as an artifact". 

But Bollum kept going. Three years later, he published details of his subsequent experiments, silencing the disbelievers, and propose that

One particularly interesting possibility concerns the use of monomers with blocked 3'-hydroxyl groups to limit the reaction to the addition of a single nucleotide residue, thereby increasing the yield of a specific product for structural analysis and further synthetic applications.

That was the discovery of TdT and the theory that it can be used to make synthetic DNA one base at a time. (Below is the only picture of Bollum I can find, from University of Kentucky. Department of Chemistry records. Gotta love the style of the 70s.)

Insight Image 5

Fast forward half a century, we now know that TdT plays a very important role in our immune system to increase the diversity of antigen receptors (reviewed here); it has also been used as a molecular biology tool to identify damaged DNA in cells (TUNEL assay) and novel RNA transcripts (RACE). 

Although our understanding of how TdT works has advanced tremendously, our ability to harness TdT, along with "blocked 3'-hydroxyl groups" to write DNA de novo has only moved at a snail's pace. 

One obstacle is that commercially available TdT is not very good at adding "blocked 3'-hydroxyl groups", likely due to the evolutionary pressure put on TdT to add natural bases in the cells. This means when scientists try to perform de novo synthesis, the first sets of experiments would likely fail. 

Many have tried, but few have persisted. At Nuclera, our instinct was to engineer TdT to work well with reversibly terminated nucleotides. After more than two years' work, we have demonstrated that engineered versions of TdT show far greater activities compared to commercially available ones when adding modified nucleotides (see our patent), thus transforming enzymatic DNA synthesis from a theoretical framework to an engineering effort. We will share more news with you very soon.

So where is Bollum now? In addition to publishing over 175 research articles, most of them on TdT, he was also in charge of a company that (still) sells TdT-related products. At the tender age of 91, I am sure he's happily retired and occasionally enjoys the annual Bollum Symposiums.

So why is enzyme better than the phosphoramidite? Stayed tuned next week.

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