Biologists have chemically-assembled DNA sequences for many years using nucleoside phosphoramidite building blocks that replicate natural bases, though achieving oligonucleotide lengths beyond 200 bases is chemically inefficient. New enzymatic strategies could circumvent those limitations, and a recent Nature article highlights advances that are being made. The template-independent DNA polymerase—terminal deoxynucleotidyl transferase (TdT)—is key to this process because it can add new nucleotides in an unbiased fashion, though to enable directed DNA synthesis as needed, the enzyme must be stopped after each step. One way to accomplish this is to tether each desired nucleotide to the enzyme itself; others are working with engineered TdT versions and modified DNA bases that can terminate a reaction and then be removed from the growing polymer to allow the cycle to repeat. The length and accuracy of enzyme-catalyzed oligonucleotide production is improving, but it does not yet outperform chemical synthesis. Because DNA synthesis is currently such a significant bottleneck in synthetic and molecular biology, advancing TdT-driven enzymatic polymerization of DNA will be a welcome breakthrough to accelerate these and other DNA-driven sciences.
Pushing the Oligonucleotide Limit: Enzymatic Non-Templated DNA Synthesis Making Gains
