Biomics

In 1955, the enzyme polynucleotide phosphorylase was discovered in the bacterium Azotobacter vinelandii, and thus the enzymatic synthesis of oligonucleotides in 2025 formally marks the 70th anniversary, and the discovery of terminal deoxynucleotidyltransferase (TdT) isolated from the calf thymus, sometimes also called the Bollum’s enzyme (by the author's surname), is 65 years old. However, it is only in the last decade that serious progress has been made in the enzymatic synthesis of extended oligonucleotides with a given sequence using this enzyme. In fairness, it should be noted that F.J.Bollum himself, back in 1986 in a review article, well ahead of his time, he described a method for such enzymatic synthesis of oligonucleotides with defined sequence. Currently, the synthesis of oligonucleotides using TdT with the desired sequence can be carried out in various ways, providing a temporary stop of polymerization after the insertion of one modified dNMP carrying a blocking group at the 3’-end, and then its resumption after the removal of the blocker, which has already been proposed a lot. Another approach is to form a complex of conventional dNTP with TdT, which leads to the incorporation of dNMP bound to TdT into the growing DNA chain, but in order to continue synthesis with a new similar complex, the enzyme from the previous conjugate must be removed from the DNA chain. Another approach is based on the competition of TdT with other enzymes, for example, with apyrase, which destroys dNTPs unused at a last stage. For effective enzymatic synthesis of oligonucleotides, it is necessary to improve the properties of TdT, including increasing its thermal stability, which is given increased attention in many studies. If for many years TdT, in addition to the calf's thymus, has also been isolated from mice, then in recent years similar enzymes have been described from birds, which, by the way, are warmer-blooded than mammals and, therefore, their enzymes are a priori more resistant to elevated temperatures. At the same time, many genetically engineered TdTs have already been created that have improved properties compared to native enzymes. There are examples of commercial custom synthesis of extended-range oligonucleotides produced by various companies (including up to 600 nucleotides), as well as a specialized "TdT synthesizer".

oligonucleotide, polynucleotide phosphorylase, terminal deoxynucleotidyl transferase, TdT, Bollum’s enzyme

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