Method makes DNA building blocks on demand

The DNA strands used in applications such as the polymerase chain reaction, DNA sequencing, and DNA data storage are typically made using nucleoside phosphoramidite building blocks. These phosphoramidites are unstable and can degrade if allowed to sit too long in a DNA synthesizer. That time is even shorter for customized phosphoramidites containing groups such as disulfides or azides, which are used for labeling DNA.

Such degradation problems could be avoided by making the building blocks as they’re needed. Kurt V. Gothelf and coworkers at Aarhus University have developed a method for synthesizing phosphoramidites on demand (Nat. Commun. 2021, DOI: 10.1038/s41467-021-22945-z). The approach could be particularly useful for labs that don’t use their DNA synthesizers daily.

In the new method, the researchers first functionalize a resin with a triazole activating group. They then immobilize a phosphitylating reagent on the resin in a flow reactor via reaction with the triazole group. To make the building blocks needed for DNA synthesis, the researchers flush nucleosides through the column, where they react with the bound phosphitylating reagent.

The researchers used the approach to synthesize various nucleoside phosphoramidites. Some of the nucleosides had sugar or backbone modifications. All of the nucleosides achieved greater than 98% yield with no need for a purification step. The researchers then used the nucleoside phosphoramidites to make DNA sequences in a DNA synthesizer.

Robert Grass, a DNA researcher at the Swiss Federal Institute of Technology (ETH) Zurich, hopes the research will enable the field to be less dependent on specialized manufacturers of DNA sequences. For example, Grass is interested in using DNA for data storage in a variety of products. In such an application, “the supply chain of phosphoramidites is very relevant,” he says. “On-demand synthesis would be highly attractive.”

Gothelf’s goal is to incorporate the phosphoramidite synthesis as a module in automated DNA synthesizers. He is in talks with companies for potential commercialization.